UniversidadPolitécnicadeMadrid
EscuelaTécnicaSuperiordeIngenieríaAgronómica,Alimentaria,ydeBiosistemas
“CaracterizaciónfuncionaldelasproteínasMTV9yMTV11ysuimplicacióneneltráficovacuolar”
TESISDOCTORAL
Doctorando:GuillermoRuanoBlanco
MásterenBiotecnologíaAgroforestal(UPM)
Madrid,2017
DepartamentodeGenéticaMoleculardePlantas(CentroNacionaldeBiotecnología,CSIC)
“CaracterizaciónfuncionaldelasproteínasMTV9yMTV11ysuimplicacióneneltráfico
vacuolar”
Doctorando:GuillermoRuanoBlanco
LicenciadoenFarmaciayenBioquímica
Directoresdetesis:Dr.DonEnriqueRojodelaViesca,Dr.DonJanZouhar
Madrid,2017
Quieroagradecera todos losmiembrosdeldepartamentodeGenéticaMoleculardePlantasdelCNB, a los compañeros y profesores del CBGP el tiempo que me han dedicado durante eldesarrollo delmáster y de esta tesis. Durante estos años vosotrosme habéis permitido darmecuenta, que la tarea ardua de investigar es emocionante y que la hora de salida demi jornadalaboralnomepreocupase.Entre todosellosquierohacermenciónespeciala JanZouharpor suamistadysudedicaciónhonestaencualquiermomentoquelerequiriese,haluchadomuchoporqueaumentasemisentidocríticoyporquemitrabajofuesemejorandocadadíaeneltranscursodeestatesis.QuieroagradeceraEnriqueconfiarenmíysuayudaalolargodetodosestosañosinclusoentiemposduros.Losproyectosenlosquehetrabajadoconélsiempremehaninteresadomucho,yhaconseguidoquenadamásverlequisiesepreguntarlesobreellos.Surigorcreoquehaconseguidoqueseaunmejorprofesionalyespero,mepermitaenel futuroseguiravanzandoanivelcientífico.Además,agradezcomuchoaJanyEnriquequeseanpersonascultasentiemposenlos que Vargas Llosa o Muñoz Molina son confundidos con nombres de bufetes de abogados.Quiero dar las gracias a Piluca, que era la primera persona que veía cuando llegaba al labo, suhumildad,susganasdeenseñarme,suagradablecharlaysucompañíaduranteestosaños.Quieroagradecer a Michi su pasión por la ciencia y haber compartido esto con él, su ayuda en ellaboratorio y su implicación y resultados en este proyecto. A Alfonso quiero agradecer sucompañía,suayudaenellaboratorioyesagraciaandaluzaqueamímeflipa.ASilvina,porhabercompartidorisasyserunaagradablecompañeraquesabedistinguirelcortedelacarneargentinoycuyalaborconMTV11esesencialparaestetrabajo.AMaite,porsupacienciayporhacermeverloimportantedelordenenestetrabajo.AMarco,porsuprofesionalidadysuexcepcionaltrabajoquemehaservidocomoreferenciaparalaredaccióndeestetrabajo.ARamón,porvenirdespuésque yo, entenderme y por lo mucho que nos hemos apoyado en el laboratorio. A Mary Paz,porqueaunquecoincidípocotiempoycompartímomentosenelcongresodePraga,estoysegurodequehubiesesidounaexcelentecompañera.AFelipe,porcontinuarunexperimentocuandoyoestuveausente.AmiscompañeroschilenosAlbertoyManuelPaneque,quehamerecidolapenapasar tiempo con ellos. A Liwen Jiang y a Jinbo Shen por su profesionalidad y porque han sidoesenciales en la progresión de este trabajo. También quiero agradecermi tiempo en el CBGP aJesúsVicenteyJoaquínMedinaduranteelmásteryalfinaldemitesis.QuieroagradeceraTomásCascón,sucapacidadparamotivarmeyapoyarmeyporhabersidocapazdeconocermemuybienyapreciarme.ACarmen,porabrirmelaspuertasdesulaboydesudespachosiemprequelohenecesitado,yporpreocuparsedequetrabajasetranquiloenmomentosdifíciles.ACarmenSimón,porsusimpatíaypornomolestarsenuncapormiscontinuasvisitasaEnrique.ARuthyYovanny,por sermaravillososcompañerosde tesisy sobretodoamigos.AGemma,por susonrisa,por sudisposición,ysucapacidadparaescucharmeyentendermisinseguridades.AAndrés,porserbuencompañeroyporsuscharlassobreciencia.AGabriel,porserungranamigoycompañeroyporhacerquemiestanciaenelCNBmerecieselapena.AMabel,porsuexcepcionalayudaconelY2H,yporsuexquisitotéquemetrajodeChina.ABárbara,porsuhumildad,subondad,suamistadyserunagrancompañeradetrabajo.ACarlosAlonsoBlanco,porsusganasdeenseñarmegenéticay suayuda con losexperimentosde floración.APilarCubas,por sudisposicióny ayuda con losensayosGUSdelaparatovasculardeestetrabajo.AVicenteyLauraporsuinestimableayudaparasabermásacercadelmutantealix queheutilizadoeneste trabajo.AMaríapor serunabuenaamiga y excepcional compañera. A Jon, por los buenos momentos durante las reunionestrasplanta.ASalomé,porsuejemploypornuncanegarseasolucionarmisdudas.AAbe,CristinaNieto,CristinaEspinosaCristinaMartínez,StellayCarlosporsergrandescompañerosydejarmeesterilizarlosmediossiemprequelonecesitaba.AAntonioLeyvaporsudisposiciónyayudaenel
desarrollodeestatesis.ÉljuntoaJavierPazAresylosmiembrosdel312mehicieronpasarmuybuenos ratos durante el almuerzo que fueronmuy necesarios para desconectar demi tarea. ACristina Navarro por su ejemplo y su amistad. A Elisa por ser una persona humilde y buenacompañera. A Marisa, Raquel y Beatriz por su contribución a este trabajo y su excelentedisposiciónhaciamis pedidospara cultivo in vitro.A Silvia por su simpatía y porque sin ella lasfotosdemicroscopíaconfocaldeestetrabajonohubieransidoposibles.AlserviciodeInvernaderoporayudarmeenelcuidadodemisplantasyenlospedidosquesolicitéduranteestatesis.Ahorametocacentrarmeenlagentedefueradelaciencia,misamigosdeFarmacia,quesiguensiendomis amigos después de tantos años, Dani, Carolina, Isa,Marta, Javi, Ana, Oihana, Irene,Natalia,Chus,Sergio,Michel,Lucía,Dave.Soisgeniales,nosayudamosmuchísimo,ycadavezquequedamosaunquenoseaconlafrecuenciaquemegustaríayconlapenadequealgunosestéislejos siempre demostramos que somos grandes compañeros de aventuras y excepcionalespersonasquecontribuísaquesealabuenapersonaquesoyyquequieroseguirsiendo.AAlbertoGorgojo,micompañerodecolegio,queaunquehayapasadomuchotiemposinvernos,hasidoungranapoyoysiemprehadichoqueteníaunamigocientífico.AJavierMuñoz,porquemesiguesqueriendoyporquenopuedepasarmástiemposinquetehagaunavisitaaHospitalet.ACristina,miamigaanestesista,quemehasapoyadocomofarmacéuticoymehasdadomuchoapoyoparacontinuarconmitesis.AClara,Óscar,MiriamÁvila,Nuria,Tamara,Coral,Mar,Miriam,Joséquemehabéis abierto losbrazos, habéis confiadoenmí yque vuestroejemplomeha servidoparacrecercomoprofesionalsanitarioymejorarlaempatíahacialospacientes.Ahoratocaescribirunaslíneasamifamilia,mispadresJesúsyPepa,porquemequeréismucho,porquenuncame faltadenada,porquemeseguísapoyandoapesarde todoparaqueseaunabuenapersona,sincerayhonesta.Porquemantenéislacabezafríaymeayudáismuchoaquenosea inmaduro ni quejica en ámbitos laborales. Ami hermano Jesús, porque sé queme quieresmucho y que aunque últimamente te vea menos y no perciba que confíes en mi desarrolloprofesionalséqueeresunexcepcionalpadreyqueRoser,mismaravillosossobrinosDaríoyElisaquehancambiadomivida, tussuegrosTerey Juan, tuscuñadosmehacensentirquesoydesufamiliaymehanacogidoconlosbrazosabiertos.TambiénamistíosGuillermoyAgueda,yamisprimasPilaryMarta,alosquequieromuchoysonmaravillosos.AmistíosPaco,MaxiySaturyamiabuelaMáxima.Porúltimo,amitíaDolores,quetesigorecordandoyhassidoungranejemplodeeleganciayvaloresqueexplicanloquesoyahoramismo.Yporúltimoati,TiffanyAnnFreda,queeltiempoquellevamosjuntosyeltiempoquenosesperaesyvaasermaravilloso.Noheconocidoanadiequemeescuche,meentiendayquemecuidecomo tú. Siempre tienesuna sonrisa, contigo siemprehayposibilidadde llegar a acuerdos, quehemoscrecidoy crecemos juntos,que ladistancianonoshaseparado,quenuestrosviajeshansidomágicos, que eres una gran profesional, que tienes el carisma que amíme falta, quemeaceptastalycómosoyyquetusamigossiemprequierensermisamigos,yquehasaceptadoamisamigosyhashechoquemequieranmás.Quequieresamispadres,yqueestánmuy felicesdesaberqueestásenmivida.Whereverweendup,wehavetocontinuefollowingourdreamsandgoalsandhelpingeachotheraswehavebeendoinguntilnow.Iwilltrymybesttoprovideyouabetterlifeandalwaysletyouknowthatthereisnothingyoucannotget.
Paraterminar,yparanoextendermemássóloquierodecir:
¡¡¡Muchísimasgracias!!!
Summary……………………………………………………………….1Resumen…………………………………………………………….…3Introduction………………………………………………………….51.Theendomembranesystemineukaryotes……………….......……………………………………………………62.Vesicletraffickingandthemachineryinvolved……………….……………………………………………………72.1.Vesicleformationmachinery.……………………………………..……………………………………………………72.2.Vesiclemovement……………………………………………………………………………………………………………142.3Vesiclefusion………………………………………………………………………………..…………………………………153.Thevacuoleinplants.Contentandphysiologicalfunctions………….……………………………………183.1.Lyticandstoragevacuoles…………...…………………………………………………………………………………193.2.Biotechnologicalpotentialofthevacuole:phytoremediationandstorage..……………………204.Vacuolartraffickinginplants.Routesandgenesinvolved…….……………………………………………214.1.Chemicalinhibitorsofvacuolartrafficking……..………………….……………………………………………214.2.Adaptorcomplexesdefinedifferentpathwaystothevacuole…………………………………………214.3.Geneexpansionforalternativepathways………..…………………………..…………………………………225.Geneticscreenstostudytraffickingtothevacuole………………………….…………………………………235.1.Geneticscreensfortraffickinggenesinyeast……….…………………………………………………………235.2.Geneticscreensfortraffickinggenesinplants…………………………………………………………………245.3.Geneticscreensfortraffickingtotheplantvacuole…………………………………………………………255.4.Screensusingchemicalgenomics………………………………………………….…………………………………27
Materialsandmethods……………………………………….291.Biologicalmaterial……………….………………………………………………………………………………………………301.1.Bacterialstrains……………….……………………………………………………………………………………………….301.2.Yeaststrains…………………….……………………………………………………………………………………………....301.3.Plantmaterial………………….………………………………………………………………………………………………..311.4Plasmids…………………………….………………………………………………………………………………………………322.Culturemethods………………….………………………………………………………………………………………………333.Methodsforbacterial,yeastandplanttransformation…….………………………………………………….374.Geneticinteractionsandphenotypicanalyses……………………………………………………………………..384.1.Arabidopsisthalianacrosses…………………………………….……………………………………………………….384.2.Floweringtimeanalyses………………………………………………….…………………………………………………384.Nucleicacidanalysisandextraction…………………………………..………………………………………………….395.Proteinanalyses……………………………………………………………….…………………………………………………..426.Microscopictechniques………………………………………………………………………………………………..………476.1.Confocalmicroscopy………………………………………………………..………………………………………………..476.2.Chemicaltreatments………………………………………………………..………………………………………………..47
Results………….……………………………………………………………481.Identificationofnovelmtvmutants…………………………………………………………….………………………...492.Mapbasedcloningofmtv9………………..……………………………………………………….………………….……..493.MTV9isaplantspecificgenewithaputativecoiled-coildomain………………….………………..……..524.MTV9expressioninRNA-seqdatasets…………………………………………………………………………………..565.MTV9promoteractivity………………………………………………………………………………………………..……….56
6.MTV9localizesprimarilyatthePVC…………………………………………………………….………………………..587.TheconservedC-terminaldomainofMTV9isresponsiblefortargetingtothePVC……..……….638.MTV9overexpressionperturbstransportofvacuolarcargobutnotPMproteins……….…………669.PVCaggregationbyMTV9overexpressionisnotaffectedbywortmannin………………….…………6910.CharacterizationofantibodiesagainstMTV9…………………………………………………………….…………7111.MTV9associateswithmembranesthatcorrespondtotheTGNandthePVC……………….………7312.MTV9andVTI11functioninseparatevacuolartraffickingpathways…………………………….……..7513.MTV11encodesahomologueofyeastVPS15…………………………………………………………….………..8014.MTV11/AtVPS15localizestoendosomalcompartments…………………………………………….………..8515.Themtv11-1mutanthasreducedPI3Plevels…………………………………………………………….…………8616.Themtv11-1mutantsshowincreasedgrowthinarsenatecontainingmedia…………….…………88
Discussion……………………………………………………………921.PhenotypicconsequencesofdisruptingMTV9activity…………………………………………………………..932.FunctionaldomainsinMTV9………………………………………………………………………………………………….943.MTV9localization…………………………………………………………………………………………………………………..964.MTV9mRNAexpression…………………………………………………………………………………………………………985.Ontheactivityofthemtv11-1allele………………………………………………………………………………………996.MTV9andMTV11,traffickingfactorsatthePVC………………………………………………………………….1017.Arsenatephytoremediation………………………………………………………………………………………………….102
Conclusions……….…………………………………………………103References…………………………………………………………..103
1
SummaryThis doctoral thesis adresses the characterization of MTV genes that are involved in vacuolar
traffickinginArabidopsis.Thevacuoleisanessentialorganelleforadaptativestrategiesofplants
andhas a great agronomic andbiotechnological importance for its storage capacityof essential
proteinsforhumannutritionorofrecombinantproteinsforbiotechnologicalpurposes.
Inthisthesis,twomutantsmodifiedintraffickingtovacuole(mtvmutants)havebeenstudiedand
pointmutationsintheMTV9/At1g24560andMTV11/At4g29380geneshavebeenidentifiedas
theresponsiblesforthedefectsinvacuolartrafficking.
The MTV9 gene is plant-specific and encodes a protein that is located at the prevacolar
compartment,possiblydirectedtothatcompartmentbythepalmitoylationofacysteinepresent
at itsC-terminalend.TheoverexpressionofMTV9provokestheaggregationof thepre-vacuolar
compartments and the delocalization of SNARE proteins, interfering with the trafficking of
proteins to vacuoles but not with the secretion of plasma membrane proteins. These results
indicatethatMTV9isessentialforthetraffickingtovacuolesandcouldbeinvolvedinanchoring
processesofvesiclesororganelleswiththeprevacuolarcompartmenttomediatetheirfusion.
The MTV11 gene encodes for the ortholog of the VPS15 protein, which is part of a
phosphatidylinositol-3-kinase (PI3K) complex, whose activity is required for all vacuolar related
traffickingroutes inyeastsandanimals.TheMTV11gene isessential inplantsand itsdisruption
causeslethality inpollen.Themutantmtv11-1 isolatedinthisthesis isahypomorphicallelethat
hasallowedtostudytheroleof thisgene inArabidopsisgrowthanddevelopment.Byusingthe
biomarker 2xFYVE we have obtained evidence that the synthesis of phosphatidylinositol-3-
phosphate issignificantlydecreased inthemutant, indicatingthat themutantallelereducesthe
activityofthePI3Kcomplex.Themutantmtv11-1hasaffectedthetraffickingofstorageproteinsin
seedsreservoirsandtherecyclingofplasmamembraneproteins.Thesedefectscauseadecrease
in growth and alterations in phyllotaxis under normal growth conditions. Moreover, different
assaystostudyresistancetoabioticstressesperformedinthemtv11-1mutant,haveshownthat
mtv11-1 plants are more tolerant to high concentrations of arsenate, a compound that is
2
sequestered in vacuoles once it is reduced to arsenite in the cytosol. This discoverymay have
important implications for the development of improved plants in phytoremediation of soils
contaminatedwitharsenic.
3
ResumenEstatesisdoctoralsecentraen lacaracterizacióndegenesMTVqueestán implicadosentráfico
vacuolarenArabidopsis.Lavacuolaesunorgánuloesencialen lasestrategiasadaptativasde las
plantas y tiene una gran importancia agronómica y biotecnológica por su capacidad de
almacenamientodeproteínasesencialesparalanutriciónhumanaodeproteínasrecombinantes
deusobiotecnológico.
Enestatesissehanestudiadodosmutantesmodificadosentráficoavacuolas(mutantesmtv)yse
hanidentificadomutacionespuntualesenlosgenesMTV9/At1g24560yMTV11/At4g29380como
lascausantesdelosdefectosentráficovacuolar.
ElgenMTV9esespecíficodeplantasycodificaunaproteínaqueselocalizaenelcompartimento
prevacuolar, posiblemente dirigida a ese compartimento por la palmitoilación de una cisteína
presente en su extremo C-terminal. La sobreexpresión deMTV9 provoca la agregación de los
compartimentosprevacuolaresyladeslocalizacióndeproteínasSNARE,interfiriendoconeltráfico
de proteínas a vacuolas pero no con la secreción de proteínas demembrana plasmática. Estos
resultados indican queMTV9 es esencial para el tráfico a vacuolas y podría estar implicado en
procesosdeanclajede vesículasuorgánulos conel compartimentoprevacuolarparamediar su
fusión.
El genMTV11 codifica para el ortólogo de la proteína VPS15, que forma parte de un complejo
fosfatidilinositol-3-kinasa (PI3K), cuya actividad es necesaria para todas las rutas de tráfico a
vacuola en levaduras y animales. El genMTV11 es esencial en plantas y su disrupción causa
letalidad en polen. El mutantemtv11-1 aislado en esta tesis es un alelo hipomórfico que ha
permitido estudiar el papel de este gen en el desarrollo de Arabidopsis. Mediante el uso del
biomarcador 2xFYVEhemosobtenido evidencias de que la síntesis de fosfatidilinositol-3-fosfato
está disminuida considerablemente en el mutante, indicando que el alelo mutante reduce la
actividaddelcomplejoPI3K.Elmutantemtv11-1tieneafectadoeltráficodeproteínasvacuolares
de reserva en semillas y el reciclaje de proteínas demembrana plasmática en la vacuola. Estos
defectosprovocanunadisminuciónenelcrecimientoyalteracionesenlafilotaxiaencondiciones
4
normales de crecimiento. Ensayos de resistencia a estreses abióticos han demostrado que las
plantasmtv11-1 sonmás tolerantes a altas concentraciones de arsenato, un compuesto que se
secuestra en vacuolas una vez es reducido a arsenito en el citosol. Este descubrimiento puede
tenerimportantesimplicacioneseneldesarrollodeplantasmejoradasparalafitoremediaciónde
sueloscontaminadosconarsénico.
5
Introduction
6
1.Theendomembranesystemineukaryotes.
ThepioneerstudiesbyGeorgeE.Paladeoninsulinsecretionwithinpancreaticcellsvisualizedthe
intricate network of endomembrane compartments comprising the secretory pathway (Palade,
1975). This network of intracellular compartments is referred to as the endomembrane system
andinplantsconsistsoftheendoplasmicreticulum(ER),theGolgiapparatus(GA),thetrans-Golgi
network/earlyendosome (TGN/EE), prevacuolar compartments (PVCs), also calledmultivesicular
bodies(MVBs),andthevacuole(Neumannetal.,2003).Theendomembranesystemofeukaryotic
cells allows spatial and temporal compartmentalization for the synthesis, sorting, delivery, and
degradation of cellular components. Possessing different compartments provides unique
environmentsforpost-translationalmodificationsandbiochemicalreactionsthatrequirespecific
conditions, such as a distinct pH. For example, the acidic pH of the vacuole/lysosome enables
degradationofproteins.
Most proteins that enter the endomembrane system do so in the ER, and then move
throughthedifferentcompartmentsuntilreachingtheirfinaldestination.Solubleproteinscontain
ahydrophobicsignalpeptideattheN-terminusthattargetsthemtothelumenoftheER(Blobel
andDobberstein,1975),andmay leavetheERoncetheyarequality-checkedforproper folding.
Transport between successive compartments can occur in three different ways: a) through a
vesicle trafficking step; b) through maturation of one compartment into another; c) through
heterotypic fusion between two consecutive compartments of the endomembrane system.
Proteins leave the ER from specific locations called ER exit sites (ERES) packed inside COPII
vesicles.COPIIvesiclesfuseatthecissideoftheGAreleasingtheircontents(BudnikandStephens,
2009). Transport along the GA occurs through cisternal maturation in a cis-to-trans direction,
coupledtobi-directionalvesicletraffickingbetweenthestacks(Orcietal.,1997).IntheGA/TGNa
major sorting event takes place that separates proteins targeted to the vacuolar pathway from
7
proteins targeted for secretion, packaging them in distinct vesicles. Secreted proteins follow a
defaultrouteandaretransportedfromtheGA/TGNinsecretoryvesiclesthatfusewiththeplasma
membrane. Soluble proteins targeted to the vacuolar pathwayhave specific sorting signals that
are recognized by vacuolar sorting receptors, which are transmembrane proteins that couple
cargorecognitioninthelumenofthecompartmenttorecruitmentofthemachineryrequiredfor
vesicle formation at the cytosolic side (Happel et al., 2004; Zouhar et al., 2010). Trafficking of
vacuolar cargo between the TGN and the PVC may occur through a vesicle trafficking step or
through maturation of the TGN into the PVC, without the proteins leaving the compartment
(Huotari andHelenius, 2011). ThePVCeventually fuseswith the vacuole, and vacuolar proteins
reach their final destination. In addition to the biosynthetic pathway to the vacuole, plasma
membrane proteins and secreted cargo are endocytosed to be recycled back to the plasma
membraneortobedegradedinthevacuole.Moreover,themachineryinvolvedinbiosyntheticor
endocytic trafficking may be recycled to prior compartments for further rounds of forward
transport.
2.Vesicletraffickingandthemachineryinvolved
Vesicle trafficking between compartments can be subdivided in three consecutive reactions: a)
cargorecruitmentandvesicleformationatthedonororganelle,mediatedbycargoreceptorsand
small GTPases that recruit coat proteins and effector proteins for vesicle budding; b) vesicle
movementalong thecytoskeletonmediatedbymotorproteins; c) fusionof thevesiclewith the
targetorganelleandcargodeliverymediatedbytetheringfactorsandSNAREproteins.
2.1.Vesicleformationmachinery
8
Foravesicletobeformed,themajorstepsinvolvedarecargorecognitionbythesortingreceptors,
coatrecruitment,membranedeformationandbuddingofthevesiclefromthedonormembrane.
• Sortingreceptors
Before anascent vesicle is formed, cargo selectionoccurs in the lumenof theorganelle,where
sorting receptors bind their cargo by recognizing sorting determinants. Sorting determinants in
plants are encoded in the amino acid sequence of the cargo proteins (Gershlick et al., 2014).
ReceptorsfunctioningintransportbetweenERandGolgiandthecorrespondingsortingmotifsare
conservedamongeukaryotes.TheCOPIIproteinsSEC23-24recognizesignalsforexportfromthe
ER (Yorimitsu et al., 2014)while the p24 cargo receptors and the ARF1GTPase are involved in
retrograde transport from the GA to the ER (Sun et al., 2007). In the TGN, vacuolar sorting
receptorsselectcargotobetransportedtothevacuole.Inplants,twofamiliesofvacuolarsorting
receptorshavebeendescribed,thevacuolarsortingreceptors(VSRs)andthereceptorhomology-
transmembrane-RINGH2 domain proteins (RMRs). Vacuolar sorting receptors (VSRs) are type 1
integralmembraneproteinsconsistingofalargelumenalN-terminaldomainresponsibleforcargo
binding, a transmembrane domain and a highly conserved cytosolic C-terminus that binds the
sorting machinery. Research performed with different vsr mutants in A. thaliana points to a
specializationwithinthisfamilyfortransportofstorageproteinsandsolubleproteinstothelytic
vacuole (LV) (Leeetal.,2013;Shimadaetal.,2003;Wangetal.,2011;Zouharetal.,2010).The
recognitionandbindingofVSRstocargoproteinsreliesonaminoacidmotifsinthecargoproteins,
whereas in mammals, cargo proteins contain mannose-6-phosphate modifications that are
recognizedbythesortingreceptors(Ghoshetal.,2003).RMRsarealsotype1integralmembrane
proteinswitha shorter lumenaldomainanda longC-terminal tail that contains aRINGdomain
(Shimada et al., 2003; Wang et al., 2011; Zouhar et al., 2010). RMRs have been localized to
9
endosomal compartments (Park et al., 2005a) but their physiological role is still controversial
(Bocock et al., 2009). AlthoughRMRs bind vacuolar cargo in vivo, no defects in trafficking have
beendescribedinmutantsofRMRgenes(Kimetal.,2005;Leeetal.,2013;Zouharetal.,2010).
• SmallGTPases
Cargo proteins bound to their sorting receptors accumulate in discrete siteswithin a particular
organelle, interacting with small GTPases that recruit cytosolic coat proteins to form a vesicle.
PlantgenomesencodesmallGTPasesfromtheRAB,RHO,ARF,andRANGTPasesubfamilies,but
noRASGTPaseshavebeen identified(Vernoudetal.,2003).TheRABandARFGTPasesregulate
the formation of vesicles on donor membranes and direct fusion with the target membrane
(Figure 1). These proteins associate with membranes in the GTP-bound form, which prevents
recognitionbytheRabchaperoneGDP-displacementinhibitor(GDI).SmallGTPasescyclebetween
active(GTP-bound)orinactive(GDP-bound)conformations,acycleregulatedbyGTPaseactivating
proteins (GAPs) and guanine nucleotide exchange factor proteins (GEFs). GAPs inactivate small
GTPasespromotingthehydrolysisofGTPandGEFsexchangeGDPbyGTPinordertorenderthem
active again.Although this basic cycle iswell-studied, it is unclearhow theseproteins associate
withaparticularmembranetoperformtheirspecificfunction(Nielsenetal.,2008).
10
Figure 1. Schematic representation of a role of small GTPases in vesicular fusion. The green objects represent two
different conformations of a small GTPase. When it is bound to GTP (green squares) the protein promotes vesicle
formation from the donormembrane andmay also direct vesicle fusionwith the acceptormembrane.GAP andGEF
proteins associate with small GTPases producing GTP binding and GTP hydrolysis respectively; and GDI, acts as a
chaperonethatpreventstheassociationofsmallGTPasesbindingtheGDPformofsmallGTPases.AdaptedfromDavid
Lambrightwebsite).
• Coatproteins
Fourtypesofcoatedvesicleshavebeendescribedinplants:clathrin-coatedvesicles(CCVs),coat
protein I (COPI)-coated vesicles, COPII-coated vesicles, and retromer coated vesicles (Figure 2).
Thesecoatsaremadeupofcytosolicproteins thatarerecruitedto thedonormembraneof the
11
nascentvesicle.Inadditiontotheirroleinshapingthevesicles,coatscontainsequencemotifsthat
recognizesortingreceptorsthatensurethatbothreceptorsandtheircargoeswillbe includedin
thenascentvesiclebeforeitsscission.Oncevesiclesarereleasedandtransportedbycytoskeletal
elements,vesiclesshedtheircoattofusewiththesubsequentcompartmentandeventuallythey
fuse with their target compartment to release their content. Interestingly, vesicles without
apparent coats forming from the Golgi or the ER and transporting vacuolar cargo have been
describedinplants(Fujietal.,2016;Wangetal.,2010),andmayrepresentalternativeroutesto
reachthevacuole.
Figure 2. Coat recruitment structure of different trafficking vesicles. The model shows adaptor proteins that are
responsible of cargo internalization; the GTPase enzymatic core, responsible for the identity and formation of the
vesicles;andtheouterlayerofthevesiclethatisdecoratedbycoatproteins.Adaptedfrom(Gurkanetal.,2006)
Adaptor protein complex!
GTPase!
b-propeller structure!
b-propeller and! a-solenoid structure!
a) COPII!
b) COPI!
c) Clathrin!
12
COPIIvesiclesare involved inanterogradeERtoGolgitrafficking.TheCOPIIcoatcomprisesfour
proteinswhicharearrangedasaninternalreceptor/cargo-bindingdimerofSEC23andSEC24and
anoutercagedimerofSEC31andSEC13(Staggetal.,2007).Theseheterodimercomplexesbind
activatedSAR1GTPase,andtogetherwithSEC23-24recruitthecargoprotein intheERexitsites
whereERmembraneisdeformedtoproducethenascentvesicle.SAR1GTPhydrolysisandrelease
ofCOPIIcoatsprecedesfusionwiththeGolgi(Yorimitsuetal.,2014).
CCVs are formed at the TGN and at the plasma membrane. Clathrin uses adaptor protein
complexes(APcomplexes)toconnectwithsortingreceptorsandtheirboundcargo.Therearefive
APcomplexesinanimalandplantcells(RobinsonandPimpl,2014).AP-1andAP-2recognizethe
conserved tyrosine motif present in sorting receptors and aid in the formation of CCVs at the
plasma membrane (AP-2) and the TGN (AP-1). AP-3 also interacts with clathrin to transport
membraneproteins to thePVCordirectly to the vacuole (Chenet al., 2011;Doreset al., 2012;
Saueretal.,2013;Zwiewkaetal.,2011).
COPI vesicles in plants are involved in retrograde transport from the Golgi to the ER and in
intercisternal trafficking between the Golgi stacks (Hwang and Robinson, 2009). The COPI coat
consistsoftwosubunits:F-COPsandB-COPs.COPIvesiclesinteractwithp24proteinstotransport
proteinssuchastheERD2receptorbacktotheER(Montesinosetal.,2014).
Retromer coated vesicles in plants are involved in retrograde transport of traffickingmachinery
(i.e: vacuolar sorting receptors) from the PVC to the TGN to perform further rounds of
anterograde transport. The retromer complex recognizes sorting receptors through the VPS26-
VPS35-VPS29 heterotrimer and together with sorting nexins bind phosphatidyl-inositol-3-
13
phosphate (PI3P), deforms the target membrane and performs the scission to form the
correspondingvesiclesforreceptorrecycling(McGoughandCullen,2011).
Uncoated vesicleswithout apparent protein cover havebeen reported tomediate trafficking of
storageproteins fromtheTGNto thevacuole inplantsand inotherorganisms (Gershlicketal.,
2014).
• Proteinsinducingmembranecurvature
Once the coat proteins have been recruited with the aid of small GTPases to the membrane
surface, themembrane needs to be physically deformed for formation and scission of vesicles.
Eachcellcompartmentpossessesaspecificmembranecomposition,mainlydifferentiatedbytheir
phosphoinositide (PtdIns) composition, which allows specific binding of proteins to deform the
membrane.PtdInsaremodifiedthroughphosphorylationatdifferentpositionsoftheinositolring
and specific forms label the different membrane compartments of the cell (Di Paolo and De
Camilli, 2006; Simonet al., 2014). Phosphatidylinositol-3-phosphate (PI3P), phosphatidylinositol-
4,5-phosphate (PI4,5P), and phosphatidylinositol-4-phosphate (PI4P) are the most abundant
PtdIns inplants (Simonetal.,2014)andtheir levelsare tightly regulatedbyvariouskinasesand
phosphatases (Balla et al., 2009; De Matteis and Godi, 2004). Analysis of overexpression and
knockoutmutants in PIP kinase/phosphatase genes have shown that some trafficking steps are
significantlyaffectedwhen thebalanceof thesePtdIns isnotproperlymaintained (Novakovaet
al.,2014;Tejosetal.,2014).SeveralproteindomainsthatbindPtdInswereidentifiedwithincoat
proteincomplexes,adaptorproteinsorotherproteins(e.g,EPSINs).Theinteractionbetweenthem
andthemembrane lipidsaid intheefficientmembranebendingandsubsequentvesiclescission
(Brett and Traub, 2006). Domains responsible for PtIns binding and for inducing membrane
curvature are the ENTH domain, ANTH domain, and BAR-domains (Zouhar and Sauer, 2014).
14
Proteinswith thesedomainsact incombinationwithcoatproteinsanddynamins todeformthe
membraneforbuddingandscissionofthevesicles(BrettandTraub,2006).PI4,5P isenrichedat
theneckofCCVwhereENTHandANTHdomainsbind,andPI3PbindingproteinssuchasFREE1or
FYVE1areresponsibleofcargorecruitmentanditsdeliveryintotheMVBs(Gaoetal.,2014).
2.2.Vesiclemovement
Thecytoskeletonservesasahighlydynamicplatformtoregulatethemovementandtheposition
ofthedifferentcellcompartments. Inplants,therearetwotypesofcytoskeletalfilaments,actin
filaments(F-actin)formedbypolymerizationofactinandmicrotubulesformedbypolymerization
of tubulin subunits. These filaments are dynamic structures capable of growth or disassembly
(Nogales, 2010). Motor proteins mediate the movement of vesicles along these cytoskeleton
tracks.Themotorproteinscontainaheaddomainresponsible forATPaseactivity thatenergizes
themovement,aneckthataidsthemovementandregulatetheATPaseactivity,andataildomain
thatbindstovesiclesororganelles(LeeandLiu,2004).Themotorproteinsattachedtotheactin
polymersarecalledmyosinsandtheonesattachedtomicrotubulesaredividedintokinesinsand
dyneins.Kinesinsmovetowardtheplusendofmicrotubulesanddyneinsmovetotheminusend.
Microtubuleandactin filamentsdisplaydifferent functionswithin thecell. In thecaseofplants,
where cells are larger and highly vacuolated, the use ofmyosinmotors for vesicle trafficking is
preferredsincetheyaremoresuitabletoreachlongerdistances.Infact,onlykinesin14familyhas
been associatedwith long distance transport in a retrogrademanner (likemammalian dyneins)
and the function of this protein depends on its interaction with actin (Jonsson et al., 2015;
Lawrenceetal.,2001;Reddy,2001;WicksteadandGull,2007;WilhelmJ.Walter,2015).Although
experimental evidence of myosin association with vesicles is lacking, the localization data of
certainplantmyosinsandtheeffectofactindisassemblysuggeststhatmyosinsareresponsiblefor
15
the movement of post-Golgi organelles (Avisar et al., 2012; Kim et al., 2005). In contrast, the
deliveryandorganizationofcellulosesynthasecomplexesintheplasmamembraneisgovernedby
corticalmicrotubulefilaments(Gutierrezetal.,2009).
2.3.Vesiclefusion
Once vesicles reach the target compartment, their membranes fuse to complete the transport
process.Thefusionmaybetransient(kissandrun)orpermanent(Giraudoetal.,2005),inwhich
case the vesicle membrane is incorporated into the target membrane. The major factors to
accomplishmembranefusionareSNAREproteins(solubleNSFAttachmentproteinreceptor),but
manyothercellularproteinsarerequiredtoensureproperfusionreactionsinvivo.
Multisubunittetheringcomplexesandhomodimericcoiledcoiltethers
Tethering factors play an essential function in membrane fusion reactions by establishing the
specificcontactsbetweenthedonorandtargetmembranecompartments(DubukeandMunson,
2016;YuandHughson,2010).ThesecontactsrelyonthefunctionalcycleofRabGTPasesandthe
shedding of the protein coat that allows the formation of a pre-fusogenic complex. The
multisubunit tethering complexes (MTCs) involved in the different trafficking steps of the
biosyntheticpathwayare:DSL1,forERtoGolgitrafficking;TRAPPIforGolgitoERtrafficking;COG,
for intra Golgi trafficking; TRAPPII and GARP for anterograde and retrograde TGN to Golgi
trafficking;CORVETforTGNtoPVCtraffickingandHOPSforPVCtovacuoletrafficking.Inaddition
toMTCs, several coiled coil proteins function as homodimeric tethering factors. In contrast to
MTCs, these homodimeric tethering factors show little sequence conservation across different
kingdoms (Kim and Bassham, 2011; Takahashi et al., 2010; Vukasinovic and Zarsky, 2016).
Unraveling the mechanisms governing the interaction between these tethering factors, the
16
arriving vesicles and the SNAREs involved in membrane fusion is key to understand how
competentfusogeniccomplexesareformedinthecell(ChiaandGleeson,2014).
SNAREandSMproteins
SNAREproteinsmediatethefusionbetweenthetargetmembraneandthevesicle,providingboth
specificityandenergytodrivethisprocess.SNAREscontainacoiled-coilregionreferredtoasthe
SNARE motif domain, which is essential for binding to other SNAREs and is widely conserved
among species (Weimbs et al., 1997).When a vesicle is tethered to a target compartment for
fusion,SNAREproteinspresentinthetwomembranesformatetramericbundleofcoiledhelices
thatbringsthemembranesclosetogether,eliminatingthewaterinterface,initiatingthemixingof
the lipids and eventually provoking the fusion between themembranes (Figure 3). It has been
shownthat theenergyreleased in the formationof thetetramericcomplex issufficient todrive
liposomal fusion (Wickner and Schekman, 2008). In vitro experiments have demonstrated that
there aremultiple combinations of SNAREs that are able to produce tetrameric bundles, but in
vivo only a few combinations lead to a successfulmembrane fusion (Varlamov et al., 2004). In
addition,ithasbeenproposedthattherearesomemembersoftheSNAREfamilythatcanactively
inhibitmembranefusionbyinteractingwithotherSNAREproteins(Bielopolskietal.,2014)orthat
a non-functional SNARE complex could assemble in vivo and inhibit the fusion process (Di
Sansebastiano,2013;Varlamovetal.,2004)
ThelocalizationofSNAREproteinsisusuallypredictedbysequenceconservationtocharacterized
SNAREsfromotherorganisms,asnoaminoacidsequencehasbeenidentifiedtolinkaparticular
SNAREprotein to its correspondingmembrane (Sanderfootetal., 2000; Scalesetal., 2000). For
instance,Qa (syntaxins) SNAREs arewell conservedbetween yeast andplants and thedifferent
typeslocalizeinanalogouscompartmentsinbothspecies.OnlyfortheSYP1SNAREclass,itcould
17
be assumed that its long transmembrane region targets this protein family to the plasma
membrane(Brandizzietal.,2002;Sanderfootetal.,2000).
SM(Sec1-Munc18)proteinsaresolubleperipheralmembraneproteinsthat interactwith
the syntaxin class of SNAREs to regulate the membrane fusion reaction (Hong and Lev, 2014).
Syntaxinsshiftbetweenopenandclosedconformationsthatarerespectively,competentandnot
competenttoformtetramericcomplexeswithotherSNARESanddrivemembranefusion.Current
evidence suggests that SM proteins may play a dual regulatory role in membrane fusion: by
bindingSNAREcomplexandcompletingfusionorbyinteractingwithsyntaxinsandstabilizingthe
closed conformation to prevent SNARE complex formation and membrane fusion. Examples of
both types of interactions have been described but a structural data about conformational
changes that allow the fusion is still missing (Archbold et al., 2014). Four major classes of SM
proteinsarepresentineukaryotes:SLY1,VPS45,VPS33andSEC1.Thesefourclassesintervenein
different trafficking stepsand togetherwithSNAREsprovide the specificity tomembrane fusion
reactionsinthecell.
Figure 3. Schematic diagram of a SNARE competent fusion complex. The R-SNARE protein necessary to form a
tetramericbundlewiththeQ-SNAREsresidingatthetargetmembraneisshowninblue.TheinteractionofSMproteins
withsyntaxinsand theotherSNAREs isalsoshown.Thesyntaxin-SM interaction isessential for thedifferentstepsof
membranefusionandthisassociationisregulatedbycalciumions.
18
3.Thevacuoleinplants.Contentandphysiologicalfunctions.
There are particularities of the plant endomembrane system that distinguish it from the
mammalianandyeastsystems,suchasthelackofanintermediatecompartmentbetweenthethe
ERandtheGolgi,thehighmotilityoftheGolgistacks,theprocessofcellplateformationandthe
presenceoflargecentralvacuoles(ContentoandBassham,2012;Dettmeretal.,2006;Kimetal.,
2005;Robinson,2014;ZouharandRojo,2009).Thepresenceoftheselargevacuolesinplantsisan
essential adaptation to the unique life style of these organisms (Rojo et al., 2001). These large
vacuolesprovideahighbufferingcapacitythatmaintainscytoplasmichomeostasisinthedifferent
environments where plants may happen to germinate. Moreover, large vacuoles allow for
energetically cheap growth which is essential for these autotrophic organisms to explore the
surroundingsfornutrients,waterandlight(ZouharandRojo,2009).Looseningofthecellwallby
enzymessuchasexpansinscoupled to thehigh turgorpressureprovidedbyvacuolesdrivescell
expansion(Cosgrove,2000;WangandRuan,2010)inaprocessthatisregulatedbyauxins(Lofke
etal.,2015).Thevacuoleisareservoirof ionsandmetabolitesandit iscrucialfordetoxification
andgeneralcellhomeostasis.Italsostoresproteinsandsolublecarbohydratesasreservesinseed
and vegetative tissues (Marty, 1999) and hydrolytic enzymes that function in recycling and
degradationofcellularcontents.Manyplantspeciescontainspecializedproteinstoragevacuoles
inseedtissues,whichaccumulatehighamountsofreserveproteinstobeusedduringgermination
and seedling establishment (Muntz, 2007). The proteins stored in seeds are an essential
agronomicalcommodityobtainedfromcropsandconstitutesthemainproteinsourceforhuman
andanimalnutrition(HermanandLarkins,1999).
19
3.1.Lyticandstoragevacuoles
It has been shown that plantsmay contain separate types of vacuoles within a single cell. For
instance, seed cells have been shown to contain both lytic vacuoles (LVs) and protein storage
vacuoles(PSVs)(Bolteetal.,2011;Frigerioetal.,2008).ThePSVisuniquetoplantsandisactually
a compoundorganellemadeupof three independentcompartments: thecrystalloidandmatrix
are separate structures that are responsible of storage protein accumulation,while the globoid
containsphyticandoxalatecrystalsassociatedwithmetalsthatprovideastableenvironmentfor
theaccumulationofcertainenzymes(Baudetal.,2008).Ithasbeensuggestedthatthegloboidis
alyticcompartmentinsidethePSV(JiangandSun,2002).Aquaporinmarkers(tonoplastproteins)
have been used successfully to show the existence of separate compartments (LV and PSV) at
some stages of development or as fused compartments (central vacuole) in the majority of
vegetative tissues (Frigerio et al., 2008). It has been proposed that the protein storage vacuole
originates from ER-derived compartments during seed maturation (Viotti, 2014). Throughout
embryo development the LV shrinks and appears as amembrane enclosed structure inside the
PSV(Bolteetal.,2011;Frigerioetal.,2008).DuringseedgerminationthePSVistransformedinto
a LV through acidification and progressive degradation of the stored proteins (Zheng and
Staehelin,2011).IthasalsobeensuggestedthattheERisthemembranesourceforLVformation
(Viotti et al., 2013). In vegetative tissues, where only a central vacuole with lytic features is
present,thepathwaysthattransportstorageproteinstothevacuolearestillfunctional(Sanmartin
et al., 2007). However, the physiological role of the PSV pathway in vegetative tissues remains
obscure.Interestingly,somepathogenesis-relatedproteins,e.g.thepathogenrelatedproteinPR5
and various lectins expressed in vegetative cells share the same sorting determinants as seed
vacuolarstorageproteinsandarelikelysortedbythesamepathwaysinvegetativetissues(Carter
etal.,2004).
20
3.2.Biotechnologicalpotentialofthevacuole:phytoremediationandstorage
Theplantvacuole is the largest compartment inplant cells,whichmakes ita suitable target for
biotechnological strategies aimed at improving the storage and the detoxification capacity of
plants.Forinstance,invegetativetissues,theoverexpressionoftonoplasttransporterstoincrease
the concentration gradient between the vacuole and the cytoplasm is one of the common
strategies to engineer plantswith properties such as heavymetal tolerance (jan Stomph et al.,
2009), salt tolerance, drought resistance (Park et al., 2005b), and accumulation of secondary
metabolites (Butelli et al., 2008). The storage capacity of PSVs, which accumulate enormous
amounts of proteins during seed maturation, makes them valuable biotechnological
compartments to store recombinant proteins. One of the major drawbacks for recombinant
protein expression in plant vacuoles is the saturation of the vacuolar trafficking capacity when
cargoproteinsareoverexpressed,whichleadstosecretionofthecargototheapoplasmbybulk-
flowmechanisms and limits the amount of protein produced (Denecke et al., 1990). A possible
solution to this problem would be to increase expression of the trafficking machinery that is
limiting for transport to the vacuole. However, the transcriptional regulation of the trafficking
genes in plants is not yet well characterized and the transcription factors involved in their
regulation remain mostly unknown. Finding those transcription factors would be crucial for
activating coordinately the expression of trafficking genes and increasing the transport capacity
(PizarroandNorambuena,2014).Inmammals,suchtranscriptionfactorsinvolvedincoordinated
activation of the trafficking machinery have been already identified (Sardiello et al., 2009),
supporting the idea that the capacity of trafficking pathways can be modulated through
transcriptionalregulation.
21
4.Vacuolartraffickinginplants.Routesandgenesinvolved
Genetic and pharmacological analysis has revealed the existence of parallel pathways for
traffickingtothevacuoleinplants.
4.1.Chemicalinhibitorsofvacuolartrafficking
Pharmacological inhibition has provided evidence for alternative trafficking pathways to the
vacuole being functional in plants. It was first shown that treatment with wortmannin, a
phosphatidylinositol 3-kinase and 4-kinase inhibitor, selectively blocked vacuolar trafficking of
barley lectin but not of sweet potato sporamin (Matsuoka et al., 1995). Wortmannin induces
homotypic fusion of the PVC, resulting in its enlargement and malfunction, which leads to
mistargeting of vacuolar cargo proteins to the apoplast (Jia et al., 2013). Brefeldin A (BFA) is a
fungaltoxinthatinhibitsBFA-sensitiveARF-GEFsandpreventsvesicleformationatdifferentsteps
of the intracellular trafficking pathways (Geldner et al., 2003). BFA treatment blocks vacuolar
traffickingpathwaysbutitdoesnotaffecttheAP-3dependentpathway,suggestingthattheAP-3
derivedvesiclesformedattheGolgibypassthePVConitswaytothevacuole(Feraruetal.,2010;
Wolfenstetteretal.,2012).
4.2.Adaptorcomplexesdefinedifferentpathwaystothevacuole
The adaptor protein complexes (AP-complexes) mediate sorting of receptors and associated
solublecargointospecificvesiclesfortargetingtotheirparticulardestination.Plantscontainfive
adaptor complexes (AP-1 to AP-5). The AP-2 complex is involved in endocytosis from the PM,
(Gadeyneetal.,2014)whileAP-1,AP-3andAP-4are involved intraffickingtothevacuole.AP-1
localizes at the TGN where it binds VSR1. Moreover, soluble vacuolar cargo sorted by VSRs is
22
abnormally secreted in ap-1 mutants, demonstrating that AP-1 is involved in their proper
transport tothevacuole (Happeletal.,2004;Parketal.,2013).Similarly, tonoplastproteinsare
mislocalizedandthevacuolebiogenesisiscompromisedinap-3mutants,suggestingthattheAP-3
complexalsofunctionsinvacuolartransport(Feraruetal.,2010;Zwiewkaetal.,2011).Recently,it
wasshownthattheAP-4complexresidesontheTGNseparatelyfromtheAP-1complex,whereit
alsobindsVSRreceptorsandisrequiredforvacuolartransportofseedglobulins(Fujietal.,2015),
suggestingthat,asisthecaseinanimalsandyeast,theAP-4pathwayisanalternativeroutetothe
canonicalAP-1dependentpathwayfortransportofvacuolarsortingreceptorsandtheircargoesto
thevacuole(Gershlicketal.,2014).
4.3.Geneexpansionforalternativepathways
Thespecialcharacteristicsoftheplantvacuolemayhaverequiredanexpansionintherepertoire
of genes involved in their biogenesis and function, including in the machinery for vacuolar
trafficking.Forinstance,ArabidopsisencodesfourQbVTISNAREs(VTI11,VTI12,VTI13andVTI14)
incontrasttothesingleVti1pproteinencodedbytheyeastgenome.Moreover,thereisevidence
thatthisexpansionhasresultedinfunctionalspecialization.VTI11residesatthePVCandformsa
SNARE complexwith SYP2 and SYP5 syntaxins,whereasVTI12 is located at the TGN in complex
with SYP4 and SYP6 syntaxins (Sanderfoot et al., 2001). Mutations in VTI11 and VTI12 affect
traffickingofdistinctvacuolarcargoes, supporting that theyact inparallel routes to thevacuole
(Sanmartinetal.,2007).
Another example of gene expansion and specialization in the vacuolar trafficking
machinery is the Rab5 GTPase subfamily. Arabidopsis encodes two conventional Rab5-type
GTPasesARA7andRHA1andaplantspecificisoform,ARA6(Becketal.,2012;Ebineetal.,2011).
In pollen tubes ARA7 and ARA6 containing vesicles are affected differently by the actin
23
depolymerizingdruglatrunculinB(LatB)(Zhangetal.,2010).ARA7andRHA1localizetothePVC
andareinvolvedinvacuolartrafficking.ARA6localizestothePVCandalsotheplasmamembrane
inacomplexwithSYP121andVAMP727,consistentwithadualfunctioninvacuolartraffickingand
in secretion. The common activator of these RAB GTPases is the guanine nucleotide exchange
factor VPS9. Interestingly, overexpression of constitutively active ARA7 but not ARA6
complementstherootgrowthdefectsofavps9mutant(Gohetal.,2007).Moreover,theara7and
therha1knockoutmutantsexacerbatethedevelopmentaldefectsofasyp22mutantwhereasthe
ara6mutantrescuesthem(Ebineetal.,2011),supportingthatARA6hasdifferentfunctionsfrom
the conventional Rab5 GTPases in plants. Moreover, by studying the MON1-CCZ complex that
functionsasaGEF forRab7GTPases,evidence for three independent routes to thevacuolehas
been provided: 1) a route that involves the maturation of RAB5 endosomes into RAB7 late
endosomes; 2) an AP-3 dependent route that does not involve RAB5 nor RAB7; 3) a RAB5-
dependent andAP-3 independent route (Cui et al., 2014; Ebine et al., 2011; Ebine et al., 2014;
Singhetal.,2014).
5.Geneticscreenstostudytraffickingtothevacuole
5.1.Geneticscreensfortraffickinggenesinyeast
ThefirstgenesencodingtraffickingmachinerywereisolatedingeneticscreensinSaccharomyces
cerevisiaeinthelate1970s.Thesecmutantsthatwereisolatedinatemperaturesensitivescreen,
showedatrestrictivetemperature(37°C)aberrantintracellularmembranousorganellesfilledwith
proteins that normally destined to the vacuole or plasmamembrane.Moreover, cell expansion
anddivisionwereaffectedbecauseofthedefectsinsecretionofcellwallremodelingenzymes.By
measuringtheactivityofapoplasticenzymessuchasinvertaseandacidphosphatase,thesec1and
24
sec2mutantswithcompromisedsecretionof theseenzymeswere isolated.Theaccumulationof
secretoryproteinsinendomembranestructuresinthesesecmutantsledtochangesincelldensity
inthemutants,whichwassubsequentlyusedtoisolatenovelmutants(Novicketal.,1980;Novick
and Schekman, 1979). In this way, sec mutants belonging to 21 complementation groupswere
found(Novicketal.,1981;Novicketal.,1980;Sataetal.,1998;SchekmanandNovick,2004).
Ascreenforvacuolarproteinsorting(vps)mutantswassetuptoidentifygenesspecifically
involved in trafficking to the vacuole. The screen searched for vps mutants that abnormally
secretedthevacuolarcarboxypeptidaseY,usingapep4leu2doublemutantbackground(Stevens
etal.,1982).Thepep4mutationpreventsprocessingandactivationofproCPYinsidethevacuole,
while the leu2mutation renders the strain auxotrophic for leucine. In the screening, amedium
containing theN-CBZ-L-phenylalanyl-L-leucine dipeptidewas used. In the vpsmutants, secreted
proCPYisprocessedbyperiplasmicpeptidasesandthencleavesthedipeptide,allowinggrowthin
Leu-media (Rothman and Stevens, 1986). 41 vps mutants were isolated that classified into six
differentphenotypicclasses(AtoF),dependingonthealterationsinmorphologyofthedifferent
endomembranecompartments(Bantaetal.,1988;Raymondetal.,1992).Anindependentscreen
forvacuolarmorphology(vam)mutantsresistanttocloroquine,whichaccumulatesinthevacuole
and provokes toxicity, yielded nine vam mutants (Wada et al., 1992). In 2002, a genome-wide
reversegeneticscreenusingacollectionof4653homozygousdiploidgenedeletionyeaststrains
andscoringforCPYsecretionbywestenblotyielded93newVPSgenes(Bonangelinoetal.,2002).
5.2.Geneticscreensfortraffickinggenesinplants
Plasma membrane transporters such as PIN1 (auxin efflux membrane protein) and tonoplast
transporterssuchasTIP2;1(vacuolaraquaporin)fusedtofluorescentproteinshavebeenusedas
markersto identifymutantswithalteredsubcellulardistribution(Avilaetal.,2003;Feraruetal.,
25
2010).PIN1isendocytosedandtheneitherrecycledbacktotheplasmamembraneortargetedto
the vacuole for degradation. In a screen of amutagenized population ofM2 plants expressing
PIN1-GFP,twopatmutants(proteinalteredtrafficking)showingPIN1accumulationinintracellular
endosomeswereisolated.Theircharacterizationrevealedthatthedefectivetraffickingwasdueto
mutations inAP-3 components, demonstrating thatAP-3was required for vacuolar targeting of
PIN1andfor lyticvacuolebiogenesis (Feraruetal.,2010;Zwiewkaetal.,2011). Inascreenofa
mutagenized population of M2 plants expressing GFP-TIP2;1 (Avila et al., 2003) , two of them
werecharacterized:themvp1mutantthatshowsalteredERmorphologyandretentionofvacuolar
and secretory proteins in the ER (Agee et al., 2010) and the csp-1 mutant that affects vacuole
morphology and cell shape (Chary et al., 2008). A screen ofmutagenized plants containing the
Golgi associated protein sialyl transferase fused toGFP (ST-GFP) led to the identification of the
gom8mutantshowinganaberrantERmorphologyduetoamissensemutationinRHD3,aGTPase
involvedinmorphogenesisoftheER(Stefanoetal.,2012).
5.3.Geneticscreensfortraffickingtotheplantvacuole
Several genetic screenshavebeendeveloped inplants specifically aimedat identifying vacuolar
traffickinggenes.Basedon theobservation thatamutant in thevacuolar sorting receptorVSR1
secreted storage proteins (12S globulins and 2S albumins) that accumulated as unprocessed
precursors(Shimadaetal.,2003),acollectionof28000T-DNAmutantswasscreenedtoidentify
mutants defective in processing of storage proteins. This rendered eight mutants, referred as
maigomutants (Lietal.,2013;Shimadaetal.,2006;Takagietal.,2013;Takahashietal.,2010),
fourofwhichhavebeenalreadycharacterized.ThefirstMAIGOgeneidentifiedwasVPS29,coding
forasubunitof the retromercomplex.ThismutantaffectsVSRrecycling fromthePVCandasa
consequencestorageproteinsaresecreted(Shimadaetal.,2006).Inadditiontothis,thegroupof
26
Dr.Hara-Nishimura developed another screen based on the increased fluorescence observed in
mutantsthatsecretetheartificialvacuolarcargoGFP-CT24,whichconsistsofGFPfusedtotheC-
terminal sortingsignalof thesoybeanstorageproteinconglycinin (Nishizawaetal.,2003).From
thisscreening,morethan100mutantsshowingagreenfluorescentseedphenotype(gfsmutants),
indicative of missorting of the transgene GFP-CT24 to the apoplast, were isolated. GFS1 was
shown to encodeVSR1,GFS2 endodedKAM2 (gfs2), amembraneprotein involved in endocytic
processes, andGFS9 encoded a protein required for anthocyanin accumulation in the vacuoles.
Thegfs9mutantshowsthatalterationsintraffickingofvacuolarproteinscanaffectthedeposition
ofothervacuolarcontent(Fujietal.,2007;Ichinoetal.,2014).
Barley lectin is a vacuolar protein that contains a C-terminal vacuolar sorting signal (Ct-
VSS) that is necessary and sufficient to target soluble endomembrane cargo into the vacuole
(Bednarek andRaikhel, 1991;Rojoet al., 2001). CLAVATA3 (CLV3) is anextracellular ligand that
activates the CLAVATA signaling pathway to restrict the size of the stem cell pool in the shoot
apicaland flowermeristemsofArabidopsis (Fletcheretal.,1999).Agenetic screen forvacuolar
traffickingmutantswassetupusingachimericfusionproteinbetweenCLV3andthebarleylectin
Ct-VSS expressed under a constitutive 35S promoter (VAC2 construct). In wild type plants the
VAC2protein (CLV3-CtVSS)accumulates in thevacuolewhere it is inactive (Rojoetal.,2001). In
mutantswheretraffickingto thevacuole is impaired,VAC2 issecretedto theapoplast,where it
reduces the pool of stem cells in the shoot apical meristem and may eventually terminate it
(Sanmartinet al., 2007).With this assay,modified trafficking to the vacuole (mtv)mutantswith
terminatedmeristemswere isolated. In apreliminary screen fourmutants (mtv1 tomtv4)were
found.MTV1encodesanepsinrelatedprotein;MTV2encodesthevacuolarsortingreceptorVSR4;
MTV3encodesthephosphoinositidephosphatasePTEN2bandMTV4encodestheARF-GAPAGD5
(Saueretal.,2013).Mutantsinsomeoftheparaloguesofthesegenesalsohadmtvphenotypes,
27
namelythevsr1,vsr3,agd12andpten2bamutants. Inaddition,reversegeneticanalysisshowed
thatmutants in the SNAREVTI12and in its SM-regulatorVPS45also secreted theVAC2protein
resultinginterminatedmeristems(Sanmartinetal.,2007;Zouharetal.,2009).
5.4.Screensusingchemicalgenomics
Thetechniquesthatcombinetheuseofchemicallibrariesofcompoundswiththeeffectofthese
compounds in vivoare referredas chemical genomics, and represent apowerful tool todissect
essentialand/orredundantcellmechanisms.Asmallmoleculecantargetallmembersofthesame
proteinfamilyandrevealaneffectthatwouldnotbedetectedifonlyonememberwasdisrupted.
Furthermore,smallmoleculescouldtargetessentialgenesthatarelethalwhenknockedout(Hicks
andRaikhel,2012).Sortinsareinhibitorsofvacuolartraffickingdiscoveredinachemicalgenomics
screenbymeasuringCPYsecretioninyeast.Someoftheisolatedsortinsweresubsequentlyshown
to alter vacuolar trafficking and/or vacuolarmorphology inplants (Zouhar et al., 2004).Genetic
screenshaveyieldedmutantshypersensitivetocertainsortinsinyeastandplants(Chandaetal.,
2009;Norambuenaetal.,2008;Rosadoetal.,2011).Inascreenforcompoundscausingdefectsin
gravitropism in Arabidopsis, 34 molecules causing altered gravitropic response and aberrant
morphologyoftheendomembranesystemwereisolated(Surpinetal.,2005;Surpinetal.,2003;
Yano et al., 2003). Another example of a small molecule derived from chemical genomics that
affects trafficking is endosidin1. Using pollen expressing GFP-RIP1 fluorescent marker and
analyzingitsgerminationinvitroledtothediscoveryofafamilyofdrugscalledendosidins.Some
ofthemcausedmislocalizationofGFP-RIP1andresultedinincreasedcytosoliclocalizationofGFP-
RIP1, inappropriate localizationat the tip,and lossofpolargrowth.RIP1 isaplasmamembrane
proteinthat interactswithROPandlocalizesatthetipofpollentubes.Theactivityofthesetwo
proteins is essential for polar growth that is necessary for pollen germination. Indeed, pollen
28
grains treated with endosidin1 are not able to grow in a polar manner, and RIP1-GFP is
mislocalizedtothecytoplasm(Robertetal.,2008).
29
MaterialsandMethods
30
1.Biologicalmaterial
1.1.Bacterialstrains
• ForpropagationofvariousplasmidstheDH5αstrainofEscherichiacoli(E.coli)wasused.
• For recombinantproteinexpression inbacterial cultures theBL21 (DE3)RosettaTM E.coli
strainwasused.
• ForpropagationandpurificationofGatewayTMplasmidscarryingtheccdBgenetheDB3.1
E.colistrainwasused.
• For transient expression in Nicotiana benthamiana and stable transformation of
Arabidopsis thaliana the C58C1 strain of Agrobacterium tumefaciens harbouring the
pGV2260plasmidwasused.
1.2.Yeaststrains
• Saccharomyces cerevisae AH109: MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ,
gal80Δ(Clontech TM).Thisstrain isused forauxotrophydependentyeast tohybrid (Y2H)
experimentsandmatingpurposeswithY187strain.
• SaccharomycescerevisaeY187:MATα,ura3-52,his3-200,ade2-101,trp1-901,leu2-3,112,
gal4Δ, met–, gal80Δ (Clontech TM). This strain is used for auxotrophy dependent Y2H
experimentsandmatingpurposeswithAH109strain.
• For yeast two-hybrid screening, we used a Y2H librarymade from cDNAs derived from
RNAisolatedfromplantsstarvedforphosphate(Pugaetal.,2014)clonedinthepGADT7-
Rec vector (Clontech TM) following theMatchmakerprotocol PT3529-1 (Clontech TM) and
tranformedintotheY187strain.
31
1.3.Plantmaterial
• Arabidopsisthaliana(L.Heynh),accessionsColumbia-0(Col-0)andLandsbergerecta(Ler).
• Nicotianabenthamianafortransientexpressionassays(Table1)
• ArabidopsisthalianatransgeniclinesinCol-0background(Table1).
• ArabidopsisthalianaT-DNAmutantallelesinCol-0background(Table2).
• Themtv9-1 andmtv11-1mutantswere isolated from an ethylmethanesulfonate (EMS)
mutagenized population derived from the L1 transgenic line expressing the VAC2
transgene, which consists of the CLV3 protein (At2g27250) fused to the barley lectin
vacuolar sorting signal expressed under 35S constitutive promoter (Rojo et al., 2002),
stably transformed in a clv3-2 mutant in Ler background (Sanmartin et al., 2007).
Therefore, the original mtv mutants obtained by forward genetics are in Ler clv3-2
background.
• Arabidopsis thaliana subcellular marker lines: wave2R (mCherry-RabF2b/ARA7), wave2Y
(YFP-ARA7), wave13R (mCherry-VTI12), wave22R (mCherry-SYP32), wave7R (mCherry-
RHA1)(Geldneretal.,2009);SYP61-CFP(Drakakakietal.,2012);VHA1-RFP(Dettmeretal.,
2006),YFP-2xFYVE(Vermeeretal.,2006).
• Subcellularmarkersusedforprotoplasttransfectionandmicroscopyanalyses:SYP41-CFP,
mRFP-VSR2, ARA7Q69L-RFP, ARA7-RFP, mRFP-SYP61, ManI-RFP (Golgi Marker), mRFP-
SYP61, Aleurain-RFP, RFP-SCAMP1 and RFP-VIT1 were used when cargo sorting was
analyzed. These subcellularmarkerswere generated at Liwen Jiang´s lab using pBI221
gatewayTMcompatibledestinationvector.
32
1.4Plasmids
-pDONR207andpDONR221GatewayTMplasmidswereusedtogenerateentryclones(Invitrogen).
An integration reaction is carried out by homologous recombination between the PCR product
carrying attB flanks (recombination site for bacteriophage λ), attB1 (5’-
GGGGACAAGTTTGTACAAAAAAGCAGGCTNN-(sequence of interest)-3’) and attB2 (5’-
GGGGACCACTTTGTACAAGAAAGCTGGGTN-(sequence of interest)-3’) and the pDONR207 or
pDONR221containing theattP sites. This results in the formationof anentry clone that canbe
recombinedintodestinationvectorscarryingattRsites.Thepositiveclonesareselectedbasedon
itsresistancetotherespectiveantibioticmarkerpresentintheplasmidused(gentamycinincase
ofpDONR207andkanamycinincaseofpDONR221).
- pGADT7: GatewayTM-compatible destination vector used in the yeast two-hybrid (Y2H)
experiments(Chinietal.,2007).Theproteinof interest isfusedupstreamtoanHAepitopeand
downstreamoftheGAL4activationdomain(AD)undercontroloftheT7promoter.
- pGBKT7: GatewayTM-compatible destination vector used in the yeast two-hybrid (Y2H)
experiments(Chinietal.,2007).TheproteinofinterestisfuseddownstreamoftheGAL4binding
domain(BD)andupstreamtoac-mycepitopeunderthecontroloftheT7promoter.
- Binary plasmids (destination vectors): pGWB3, pGWB5, pGWB6, pGWB14 (Nakagawa et al.,
2007), andp-UBQ10-N-RFP (Grefenet al., 2010).pGWBsareGatewayTM-compatible vectors and
presenthygromycinandkanamycinresistantgenes.pGWB5andpGWB6allowtranslationalfusion
of a protein of interest with GFP at the C-terminus and the N-terminus, respectively. The
recombinantproteinexpressionisdrivenbythe35Sconstitutivepromoter.IncaseofpGWB3the
promoterofthegeneofinterestisfusedtotheβ-glucuronidasegene(GUSgene).Thedestination
vectorpGWB14carries the35Sconstitutivepromoter in framewith thegeneof interest tagged
withthreeconstitutiveHApolypeptides.
33
-pBI221modifiedplasmidwasusedforprotoplasttransformation(Gaoetal.,2014).Thisplasmid
carries an Ampicillin resistant gene and allows LR GatewayTM recombination reaction using
Gatewaycompatibleentryvectors(collaborationwithJinboShenatLiwenJiang´slab,CUHK).
- Plasmid for recombinant protein expression: pDEST17 (GatewayTM plasmid, Invitrogen). This
plasmid carries a T7 based promoter, recognized by the T7 polymerase. The T7 polymerase is
encoded by the BL21 (DE3) bacterial genome and its expression is inducible by IPTG. The BL21
(DE3)rosettastrainusedforexpressionisoptimizedforthecodonusageofeukaryoticgenes.The
resultingrecombinantprotein is taggedwithsixhistidinesat itsC-terminalpart toallowprotein
detectionandpurification.
-Plasmid for inducibleexpression inplants:pMDC7,aGatewayTM-compatibledestinationvector
for estradiol-induced expression of the protein of interest in planta (Curtis and Grossniklaus,
2003).
2.Culturemethods
• Bacterialculturemethods
ThedifferentEscherichiacolistrainswereincubatedovernightat37°CinLuriaBertani(LB)media
with appropriate antibiotics. LBmedia ismade of bacto-tryptone (10 g/l), yeast extract (5 g/l),
sodiumchloride(10g/l)andforcultureinsolidmedia,agarwasadded(3%,18g/l).Theworking
concentrations for thedifferentantibioticswere:ampicillin (100μg/mL),gentamicin (50μg/mL),
hygromycin(40μg/mL),kanamycin(50μg/mL),rifampicin(50μg/mL),spectinomycin(50μg/mL),
streptomycin(10μg/mL)andtetracycline(5μg/mL).
ForproteinexpressioninE.coli,coloniesweregrowninliquidLBuntilreachinganopticaldensity
of 0.6, induced with 0.4 mM IPTG, and aliquots were taken for protein extraction at different
times.
34
The strains ofAgrobacterium tumefaciens C58C1 were incubated during 2 days at 28°C in YEP
media (10 g bacto-peptone, 5 g of NaCl, and 10 g of yeast extract per liter) with appropriate
antibiotics.
In order to preserve E.coli andA. tumefaciens transformant lines during short periods of time,
strainswerekeptat4°CinsolidLBmediawithadequateantibiotics.Forlongerperiodsoftimethe
cellswereresuspendedina20%glycerolsolutionandkeptat–80°C.
• Yeastculturemethods
TheAH109andY187strainsofS.cerevisiaeweregrowninYPADandYPDmedia,respectively
(providedbyClontechTM).TheliquidYPADmediumisastandardrichYPDmedium(20g/L
peptone/tryptone,10g/Lyeastextract,20g/LglucosewithpHadjustedto5.8)supplemented
with40mg/Ladeninetoallowgrowthofstrainscarryingtheade2mutation.Thecorresponding
solidmediawassupplementedwith20g/Lofbactoagar(DuchefaTM).Transformationofboth
AH109andY187strainswithpGADT7andpGBKT7plasmidsledtonormalgrowthofyeastcellson
SDmedium.TheSDmedium (syntheticallydefinedmedium)isminimalmediathatisroutinely
usedforculturingS.cerevisiae.SDbasesupplieseverythingthatayeastcellneedstosurvive
35
(including20g/Lofglucoseand6,7g/Lofyeastextractascarbonsourcesandnitrogensources,
respectively)anditissupplementedwiththeaminoaciddrop-out(DO)mixanditspHadjustedto
5.8withKOH.Inaddition,20g/LbactoagarwereaddedfortheSDsolidmedia.
SD-WL: The SD media without tryptophan and leucine, prepared with the -Trp/-Leu DO
Supplement(Clontech,No.630417).
SD-WLH:TheSDmediawithouttryptophan,leucineandhistidine,preparedwiththe-Leu/-Trp/-
His/DOSupplement(Clontech,No.630419).
SD-WLA:TheSDmediawithout tryptophan, leucineandadenine,preparedwith the -Ade/-His/-
Leu/-TrpDOSupplement(Clontech,No.630428)andsupplementedwith0.002%histidine.
SD-WLHA:TheSDmediawithouttryptophan, leucine,histidineandadenine,preparedwiththe-
Ade/-His/-Leu/-TrpDOSupplement(Clontech,No.630428).
3-AT (3-amino-1,2,4-triazol): this compound is a competitive inhibitor for histidine biosynthesis
and it is added in case of bait autoactivation or to assess the strength of protein-protein
interactions in the yeast two hybrid assays, which use histidine auxotrophy as a marker for
protein-proteininteractions.
• Arabidopsisinvitroculture
Arabidopsis seeds were surface sterilized with a mixture of 70% ethanol and 0,05% Tween-20
during10minutes.Afterthistime,seedswerewashed3timeswithsterilizedwaterandwerecold
treatedforuniformgerminationduring3daysat4°Cindarkness.Seedsweregerminatedon½MS
media(MurashigeandSkoogmedia,DuchefaTM)supplementedwith1%sucrose,and0,7%agaror
1,2% for horizontal or vertical growth, respectively. The growth chamberswere set to long day
photoperiodconditions(16hoursoflightand8hoursofdarkness)witha60%relativehumidity.
36
• Assaysinarseniclimitingconditions
Seedsweresurfacesterilizedandcoldtreatedduring2daysat4°Cindarkness.Seedsweresown
onto 1% agar vertical plates containing low phosphate Johnson media (12,5 µM phosphate)
(Johnson CM, 1957) and grown for five days. Subsequently, seedlings were transferred to low
phosphateJohnsonmediasupplementedwithorwithout10or20µMarsenateandrootgrowth
inthenewplateswasmeasured3daysaftertransferringtheplants.
• CultivationofArabidopsisthalianainsoil
Tendaysoldseedlingsweretransplantedintoamixofsoilandvermiculite(ratio3:1).Plantswere
growninthegreenhouseat22°Cwithaphotoperiodof16hoursoflightand8hoursofdarkness
forlong-dayconditions,and8hoursoflightand16hoursofdarknessforshort-dayconditions.
3.Methodsforbacterial,yeastandplanttransformation
• Bacterialtransformation
Transformation of competent DB3.1 and DH5α E. coli strains was carried out by heat-shock
protocolsaspreviouslydescribed(Sambrook,1989).CompetentE.colicellswerepreparedusinga
calciumchloridetreatment(Hanahan,1985).TransformationofcompetentA.tumefaciensC58C1
strain was carried out as described in (Weigel, 2002). Competent Agrobacterium cells were
generatedusingafreezingmethodandcalciumchloride(Holstersetal.,1978).TransformedE.coli
andA.tumefaciensstrainswereplatedonselectivemedia(LBwithcorrespondingantibiotics)and
incubatedovernightat37°Cor48hoursat28°C,respectively.
37
• TransformationofArabidopsisthaliana
A. thaliana plants were grown in soil during 20-25 days in long-day conditions before their
transformation. Young inflorescences were immersed in a suspension of A. tumefaciens
transformedwith the construct of interest resuspended inMurashige and Skoog (MS)medium
supplemented with 5 % of sucrose, 0.02 % of Silwet L77 (a surfactant agent) and 50 mM of
benzylaminopurine(BAP,acytokinin)for10minutes(Bechtold,1993).Afterinmersionplantswere
setinatraycoveredwithplasticfor2daysandthentheplasticwasremovedandplantswerekept
inthegreenhouseuntilseedswereharvested.ThecollectedseedsweresowninMSmediumwith
thecorrespondingantibioticsforselectionoftheresistanttransformants.
• AgroinfiltrationofNicotianabenthamianaleaves
Three to four week old N. benthamiana plants were used for transient transformation
experiments. Bacterial suspensions of A. tumefaciens transformed with the corresponding
constructswere used for infiltration of the abaxial side ofN. benthamiana leaves as previously
described (Sparkes et al., 2006). To enhance the expression of a protein of interest, the leaves
wereco-transformedwithAgrobacteriumcarryingthepBin61-P19plasmidthatencodesthep19
proteinoftomatobushystuntvirus,asuppressorofpost-transcriptionalgenesilencing(Voinnet,
2003).After2or3daysofinfiltration,leaveswereanalyzedbyconfocalmicroscopy.
• Saccharomycescerevisiaetransformation
S. cerevisiae cellswere transformed as described inMatchmakerGal4 Two-HybridUserManual
(ClontechTM).Briefly,culturedyeastcellsareincubatedovernightandthenrefreshedbydilutionin
freshmediaandsubsequentgrowthuntilreachinga0.6-0.7ODvalue,followedbyapolyethylene
38
glycol(PEG)/LiAc-basedpreparationofcompetentyeastcells.Forplasmidtransformationweused
aheatshockbasedprotocolwhereyeastcellsareincubatedat30°Cfor30min,mixedwithDMSO
(5%volume)andincubatedfor15minat42°Cbeforeplatingontoselectivemedia.
4.Geneticinteractionsandphenotypicanalyses
• Arabidopsisthalianacrosses
Recipientflowersatstage13(whenthepetalswerebarelyvisible)wereemasculatedwithfine
forcepsandpollinizedbygentlybrushingthematureanthersofdonorplantontothestigma.
• Floweringtimeanalyses
To quantify the differences in flowering time between mtv9-2 and Col-0, three independent
experimentswerecarriedout.Rosetteandcaulineleaveswerecountedatthetimeofflowering,
consideredas the timewhenthe first floweredopenedandpetalswerevisible.A t-student test
wasusedforstatisticalcomparisonofthemeanfloweringtime(countedasnumberofleaves)in
mtv9-2andCol-0.
4.Nucleicacidanalysisandextraction
• ExtractionofplasmidDNAfrombacteria
Toisolateplasmids,weusedtheWizardPlusSVMiniprepsDNAPurificationSystem(PromegaTM).
• PlantDNAisolation
For standard PCR analyses, A. thaliana genomic DNA was extracted following an isopropanol-
based method described previously (Doyle, 1990). DNA samples intended for whole genome
sequencingwere prepared using the plant DNeasy kit (QIAGENTM). Thismethod ensures longer
39
DNA fragments and allows successful library preparation for the next generation sequencing
(NGS).Incaseofmtv9-1,onegramofyoungleavescollectedfromtwentyfiveplantsshowingthe
mtvphenotype(meristemterminated)wereusedforDNAisolationandtheNGS.
• Gel/PCRDNAfragmentsextractionkit
DNAfragmentsseparatedbyagarosegelelectrophoresisorDNAamplifiedinapolymerasechain
reactions(PCR),werepurifiedusingQIAquickGelExtractionKit(QIAGEN).
• ExtractionofplasmidDNAfromyeast
To isolate plasmids from yeast, we utilized previously described methodology (Hoffman and
Winston,1987).Forsequencingtheplasmids,wereretransformedthemintothestandardDH5α
E.colistrainandsubsequentlyisolatedbyamethoddescribedinthesection4.1.
• AmplificationofDNAfragments
Taq polymerase (Roche)was used for standardPCR amplifications and thehigh-fidelity Phusion
DNA polymerase (Roche) for cloning purposes. Primers used for PCR amplifications throughout
thisworkarelistedintheTables2and3.
40
41
42
• Gelelectrophoresisofnucleicacids
DNA fragments obtained by PCR or the fragment analysis derived from plasmid digestionwere
visualisedbyagarosegelelectrophoresisusingethidiumbromidein1xTBEbuffer(50mMTris,1
mMEDTA,pH8).
• SequenceanalysisofDNA
LibrarypreparationandtheNGSsequencingoftheDNAfromthepoolofmtv9-1plantswasdone
by the Genome Center at the Max Planck Institute for Developmental Biology. Reads were
mappedontotheTAIR10genomeandaregionthathadonlyreadsfromLandsbergaccesionwas
identified.Within this region, EMS type polymorphisms (G/A or C/T) producing changes in the
open reading framewere selected.For sequencealignmentofMTV9andSTV9 theGENOMATIX
programalignmenttoolwasused(http://www.genomatix.de/cgi-bin/dialign/dialign.pl).
5.Proteinanalyses
• SDS-PAGEproteinanalyses
ArabidopsisthalianaandNicotianabenthamianaplantsampleswerefrozeninliquidnitrogenand
homogenized in Laemmli loading buffer (Laemmli, 1970). The samples were boiled during ten
minutes and after a short centrifugation to pellet cell debris theywere loaded on the gels. To
visualizeseedstorageproteins,gelswerestainedwithCoomassieBrilliantBlue.Forwesternblot
analysis, proteins were transferred onto ECL-nitrocellulose membranes (GE Healthcare Life
Sciences).Themembraneswerefirstincubatedinblockingsolution(5%non-fatmilkinTBS+0.1%
Tween-20)toavoidunspecificproteinbinding,andthenincubatedwithprimaryantibodiesdiluted
in 1% non-fat milk in TBS+0.1% Tween-20, washed five times in TBS+0.1% Tween-20 buffer,
incubatedwithsecondaryantibodiesfusedtohorseradishperoxidasedilutedin1%non-fatmilkin
43
TBS+0.1% Tween-20, washed five times in TBS+0.1% Tween-20 buffer, incubated with ECL
reagentsandexposedonfilmsfordetectionoftheproteins.
• ExpressionofaMTV9fragmentforantigenproduction
Weexpressed aN-terminal fragment of theMTV9protein (N-terCORE) inE.coli. Analysis of the
distribution in soluble and insoluble fractions showed that the protein was mostly found in
inclusion bodies. 50 mL of a bacterial culture from a colony that overexpressed the MTV9
fragmentwereresuspendedin5mL50mMTRISbuffersolutioncontaining2mMEDTAatpH=8.2
andlysedbyultrasoundwithasonicatingprobe,centrifugedat13,000rpmfor15minutesat4°C
and the resultingpelletwas resuspended inLaemmli loadingbufferandsubjected toSDS-PAGE.
ThegelbandcontainingtherecombinantMTV9fragmentwascutandusedtoimmunizerabbits.
• Antibodyproductionandpurification
OncewewereabletoinduceasufficientamountoftheN-terCOREMTV9fragment,wesentthe
gel band corresponding to this fragment for antibody production. Five different rabbits from
PinedaAntikörperService(http://www.pineda-abservice.de/main.php)werepretestedforlackof
cross-reaction with Arabidopsis proteins. After initial immunization and three boost shots we
obtained a serum that recognized the endogenousMTV9 protein and was utilized for western
blotting analyses. We further purified the serum by binding to MTV9 protein transferred into
nitrocellulosemembranes and used it for immunoelectronmicroscopy analyses. Briefly, the N-
terCORE fragmentwas run and transferred to anECLnitrocellulosemembraneand thebandof
interestwascutout.A0.1MglycinepH=2,5solutionwasusedtoremovepoorlyboundproteins
during 5 min and the membrane was washed two times with 1xTBS 0,1% Tween buffer. The
membranewasblockedwith3%BSA1xTBSsolutionduring1hour,andafterwashing500µLof
44
MTV9 antibodywere allowed to bind themembrane during 3 hours at room temperature. The
supernatantwassavedandthemembranewas incubatedwith500µLof theglycinesolutionto
elutetheMTV9purifiedantibody.After10mintheglycinesolutionwasneutralizedwith23µLof
TRIS1MpH=8andthiswasthesampleusedforMTV9immunoelectronmicroscopyanalyses.
• AnalysesofArabidopsisapoplasticfluid
ToisolatetheproteincontentoftheArabidopsisapoplast,onegramof5weekoldrosetteleaves
grown in short day conditions was used. These leaves were vacuum infiltrated with 50 mL of
sodiumphosphatebuffer,pH8.0,containing150mMNaClfor15min,breakingdownthevacuum
every3min.After infiltration, leaveswerecarefullydriedwithatissuepaperandtheapoplastic
fluidwascollectedbylow-speedcentrifugation(900xg).Theisolatedapoplasticfluidwasmixed
withLaemmliloadingbufferandprocessedasdescribedinthesection5.1.
• Sucrosedensitygradients
Toanalyzelocalizationofvariousproteinmarkerstodistinctendomembranepopulations,protein
samplesfromflowertissuesor10daysoldseedlingswereseparatedonsucrosedensitygradients.
The samples (≈1-1.5 g) were lysed in 8 ml of 50 mM Hepes-KOH, pH 6.5/5 mM EDTA/13.7%
(wt/vol) sucrose/0.1 mM phenylmethylsulfonyl fluoride/1 mM DTT/ 1x Complete Protease
Inhibitor Cocktail (Roche), centrifuged (1,000g and 4 °C for 10 min) and the supernatant was
collected. 1.5 mL of the supernatant were layered on top of the 9-mL line step gradient, as
previouslydescribed(Sanderfootetal.,1998).Gradientswerecentrifugedfor2hat100,000xgin
aBeckmanSW40Tirotorat4°C.Subsequently,0.5mLfractionswerecollectedfromthetopofthe
tubeandtheproteinswereprecipitatedin10%trichloroaceticacid(TCA),washedin90%acetone
45
and dried. The protein pellets were resuspended in the Laemmli loading buffer, processed as
described in the SDS-PAGE analysis section, subjected to electrophoresis followed by
immunoblotting.
• DifferentialcentrifugationofArabidopsismicrosomalfractions
Arabidopsis seedlings were grown in the liquidMS for 10 days under long-day conditions. The
plants (≈1-1.5 g)were carefully driedwith a tissuepaper andhomogenized in extractionbuffer
(100mMTris-HCl, pH7.5, 400mMsucrose, 1mMEDTA, 0.1mMPMSF, 1XCompleteProtease
InhibitorCocktail) usingamortar andpestle, followedby centrifugationat1,000xg for15min
resulting inapellet(P1)andacorrespondingsupernatant(S1)(BasshamandRaikhel,1998).The
S1supernatantwascentrifugedat3,000xgfor15mintoproduceamembranepellet(P3).TheS3
supernantant was subsequently centrifuged at 16,000 x g for 15 min to produce a membrane
pellet (P16). 100,000 x g for 30 min to yield a membrane pellet (P100) and a soluble fraction
(S100).PelletsandsupernatantswereresuspendedintheLaemmliextractionbufferandanalyzed
bySDS-PAGEandimmunoblotting.
• ExtractionofMTV9frommembranes
1-1.5gramsofArabidopsisseedlingswereprocessedasdescribedintheprevioussectiontoyield
theS1fraction,whichwasdirectlysubjectedto100,000xgultracentrifugation.TheisolatedP100
membrane pellets were resuspended in 200 µL of the HE extraction buffer containing 0.1 M
Na2CO3,1MNaCl,2Murea,or1%(v/v)TritonX-100,respectively,andincubatedfor2honice.
Sampleswere subjected toa secondultracentrifugationat100,000xgand the resultingpellets
wereresuspendedintheLaemmli loadingbuffer.Supernatantswereprecipitatedusing10%TCA
andalsoresuspendedintheLaemmliloadingbufferandprocessedasinthe5.1.section.Samples
46
weresubsequentlyanalyzedbySDS-PAGEandimmunoblotting.
• Co-immunoprecipitationanalyses
MicrosomesobtainedfromArabidopsisseedlingswerehomogenizedin500μLofextractionbuffer
(100mMTris-HCl, pH7.5, 400mMsucrose, 1mMEDTA, 0.1mMPMSF, 1XCompleteProtease
Inhibitor Cocktail), 0.2%NP-40,and 1% bovine serum albumin (BSA). Extractswere centrifuged
twiceduring1minuteat13,000 rpmat4°C. Supernatantsweremixedwith15μLofanti-MTV9
antibodyandincubatedfortwohoursat4°C.Inanewtesttubeinwhich150μLofproteinAresin
(Roche)wereaddedandtheproteinextractwasgentlyshakenfor2hoursat4°C.Subsequently,
thetubeswerecentrifugedat13,000rpmfor30seconds.Theresinwaswashedthreetimeswith
theextractionbuffersupplementedwith1%BSA,andalsowithHEbufferlackingBSA.Afterwards,
theresinwasresuspendedintheLaemmliloadingbufferandboiledfor10minutes.Sampleswere
analyzedbySDS-PAGEandimmunoblotting.
• Antibodies
Different primary antibodies were used and these were diluted 1:1000 but express indication:
anti-GFP,anti-MTV9,anti-Aleurain(Ahmedetal.,2000);anti-CPY(Rojoetal.,2003);anti-SYP21(da
Silva Conceicao et al., 1997), anti-VSR(Sohn et al., 2003), anti-VTI11, anti-VTI12 (Surpin et al.,
2003), anti-SEC12 (Bar-Peled and Raikhel, 1997) , anti-PR5 (Palacin et al., 2010), anti-VacPerox
(Sanmartin et al., 2007), anti-TGG1 (Ueda et al., 2006), anti-BiP (Santa Cruz Biotechnology sc-
33757). Secondary antibodies fused to horseradish peroxidase were used at 1:10000 dilutions
(anti-rabbit,anti-goat,anti-mouse).
47
6.Microscopictechniques
6.1.Confocalmicroscopy
Confocal images were acquired by Leica TCS SP2 and Leica TCS SP5 multispectral confocal
microscopes (LeicaMicrosystems)witha63xwater-immersionobjective, recording images from
laser lines of 405, 488, and 561 nmwavelengths. LAS AF v.2.3.6 software was used for image
compilation.Imageprocessingincludingcolocalizationofsinglecolorprotoplastimagesweredone
with ImageJ and Photoshop (http://www.macbiophotonics.ca/imagej/). A. thaliana seedlings
visualized inconfocal imagingweregrownfor3-6daysat22°Cwitha60%ofhumidityand16-
hours photoperiod with a fluorescent light of a 100 μmol m-2s-1 intensity. We visualized root
epidermalcellsinA.thalianaandepidermalcellsoftheabaxialsideofleavesinN.benthamiana
plants.
6.2.Chemicaltreatments
A.thalianaseedlingsandcultureprotoplastsweretreatedchemicallywithBFAandwortmanninto
checkpossibledefectsintheendomembranesystemandincargotrafficking.BFAandwortmannin
weredilutedinDMSOat50µMand33µM,respectively.Seedlingswereincubatedfor2hoursin
theseconcentrationsbeforemicroscopeexamination.
48
Results
49
1.Identificationofnovelmtvmutants
SeedsfromtheL1transgenic linecontainingasingleVAC2insertioninchromosome3(17.1Mb)
weremutagenizedwithEMSandsownin48traysthatwerecollectedseparatelyin48poolsofM2
seeds.M2plantswere screened formutants showing prematurely terminated shoot apical and
flowermeristems. Inapartialscreenofsomeofthepools,eightmutants(mtv1-mtv4andmtv8-
mtv11) with prematurely terminated vegetative, inflorescence and flower meristems (mtv
phenotype)wereisolated.Usingthissamestrategy,threeothermutants(mtv5-mtv7)havebeen
identifiedandcharacterizedinthelaboratoryofDr.NatashaRaikhel(Rosadoetal.,2011;Sohnet
al., 2007). The mutations responsible for the mtv phenotype in four of these mutants were
previously identified: mtv1/At3g16270 (pool 40); mtv2/vsr4/At2g14720 (pool 43);
mtv3/pten2a/At3g19420(pool37)andmtv4/agd5/At5g54310(pool46)(Saueretal.,2013;Zouhar
et al., 2010); PhD Thesis of María Otilia Delgadillo). In this work, we characterized two other
mutants from that initial screen, mtv9 (pool 31) and mtv11 (pool 32) and identified the
corresponding genes through map-based cloning. Recently, in the lab we have carried out a
saturatingscreenofapproximately2000M2plantsfromeachofthepoolsandisolated121new
putativemutantswithastrongmtvphenotype.Wehaveidentifiedbynextgenerationsequencing
thecausativemutationof20ofthesemtvmutants,andfoundanadditionalalleleofmtv9 (pool
48).
2.MAP-basedcloningofmtv9
Themtv9-1mutantwas isolated frompool 31. The F1plants froma cross between themtv9-1
mutantandCol-0hadawild typephenotype.Moreover, theproportionofplantswitha strong
mtv phenotype in the F2mapping population (157 out of 2011 plants)was consistentwith the
expected ratio fora recessivemutationunlinked to theVAC2 transgeneproducinga strongmtv
50
phenotypewhen both themutation and the VAC2 transgene were in homozygosis. Hence, for
rough mapping of themtv9-1 mutation, we performed bulk-segregant analysis with F2 plants
displayingthemtvphenotype.WeisolatedDNAfromapoolof49plantswithmtvphenotypeand
scanned the five chromosomes for regions showinganenrichment in Ler SSLPs. In thisway,we
found that the AthZFPG SSLP marker (chromosome I) was highly enriched for the Ler
polymorphisminthepoolofmutantDNA.Genotypingofthe49individualmutantplantsrevealed
that 97of the98 chromosomeshad theAthZFPG Ler SSLP, indicating close linkage to themtv9
mutation.Moreover,theanalysisoftheseplantswithmarkersaroundAthZFPGdelimiteda1Mb
region containing the mutation. We sequenced the candidate genes in that region
VTI12/At1g26670, ISTL2/At1g25420,andTNO1/At1g24460 thatencodeproteinswithpreviously
reportedrolesinvesiculartrafficking(KimandBassham,2011),butnomutationswereidentified
in those genes. Finemapping restricted thepositionofMTV9 to 0.1Mb interval containing 34-
genes (At1g24320–At1g24640) between the BOF316 marker and the AthZPFG SSLP markers
(Figure 1A). However, no obvious candidates were apparent in that interval, so we performed
deep sequencing of a DNA sample from a pool of 30 plants withmtv phenotype from the F2
mappingpopulation(Figure1B). Inthe intervaldefinedthroughfine-mapping,wefoundasingle
EMS(G/A)mutationthatgeneratedaprematurestopcodoninthefirstexonofAt1g24560,agene
ofunknownfunction(Figure5A).ThisEMSmutantallelewasnamedmtv9-1.
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Figure1.mtv9 identificationthroughmap-basedcloning. (A)mtv9mappedtochromosome1 inan intervalbetween
BOF316(8,63Mb;3recombinantchromosomes)andAthZPG(8,73Mb;1recombinantchromosome)SSLPmarkers.(B)
TheDNAfromapoolof30plantswithmtvphenotypewasdeepsequencedandthepercentageofreadswithCol-0or
LerSNPsalongchromosome1 isshownastheblack line.Thered linemarksequalpercentageofLerandCol-0SNPs,
abovetheredlinemarksenrichmentinCol-0SNPsandbelowthelineenrichmentinLerSNPs.Theregionencircledhas
readswith100%LerSNPs.
In order to confirm that the mutation of At1g24560 is responsible for the observed mtv
phenotype,twoadditionalT-DNAmutantalleleswereanalyzed.Themtv9-2allele(SAIL_24_C10)
has a T-DNA insertion in the first exon of the At1g24560 gene and the mtv9-3 mutant
(SAIL_670_H06) has a T-DNA insertion in the fourth intron (Figure 5A). Plants homozygous for
these T-DNA insertions were isolated and crossed with VAC2 line. The analysis of the two F2
populations showed thatplantshomozygous formtv9-2 ormtv9-3 andat leastone copyof the
VAC2 transgenehadterminatedmeristems, reproducingthephenotypeofmtv9-1plants (Figure
5B).Moreover,we identified an additionalmtv9 allele in anmtvmutant isolated frompool 48,
whichcontainedanEMSmutationthatdisruptedthespliceacceptorsiteofthethirdintronofthe
MTV9 gene (mtv9-4 allele). Altogether, the analysis of these four independent alleles strongly
supports thatMTV9 is required forvacuolar traffickingofVAC2andthateliminating its function
resultsinVAC2secretionandconsequentprematuremeristemtermination.
52
Figure5. Independentmutantalleles forAt1g24560showmtvphenotype in thepresenceofVAC2. (A)Aschematic
representationoftheMTV9genelocus,showingthepositionsofthefourisolatedmtv9mutantalleles.Themtv9-1and
mtv9-4areEMS-inducedpointmutantswhilethemtv9-2andmtv9-3areT-DNAinsertionalmutants.(B)Confirmationof
meristemterminationinmtv9-2andmtv9-3mutantsexpressingtheVAC2transgene.
3.MTV9isaplantspecificgenewithaputativecoiled-coildomain.
Asearchforhomologousproteinsthroughsequencealignmentsearchtools(Altschuletal.,1990;
GishandStates,1993)showedthattherearenohomologuesofMTV9presentoutsidetheplant
kingdom. In contrast, close homologues of MTV9 were found in all land plants, including in
Selaginella moellendorffii, Physcomitrella patens andMarchantia polymorpha. The Arabidopsis
genomehasalsoaparalogousgene(At3g49055)thatwenamedSTV9. Interestingly,seedplants
contain bothMTV9 and STV9 paralogues whereas non-seed plants like S. moellendorffii and P.
patensonlycontaintheMTV9paralogue(Figure6).
53
Figure6.PhylogeneticanalysisofMTV9homologuesinArabidopsis,rice,PhyscomitrellaandSelaginella.
The Arabidopsis STV9 protein has 27% overall identity (46% similarity) withMTV9, and
homology is maximal in the C-terminal half (Figure 7). We analyzed whether STV9 was also
involved in traffickingofVAC2bycrossing the transgene intoT-DNAknockoutmutants forSTV9
gene (SAIL_193_D05 and SALK_029261).However, thehomozygous stv9mutant did not display
meristemterminationinthepresenceoftheVAC2transgene,indicatingthatSTV9hasadifferent
functionthanMTV9.
54
Figure7.AlignmentofArabidopsisMTV9andSTV9proteinsequences.Redcolorindicatessequenceidentityandblue
colorindicatessequencesimilarity.
Structural sequence analysis showed thatMTV9 has no previously described functional
domain (Wang et al., 2007). However, the Pfam server predicted MTV9 to contain coiled-coil
55
domainsalongitssequence(Finnetal.,2016)(Figure8).Thisisconsistentwithapriorprediction
ofMTV9beingacoiled-coilprotein(Gardineretal.,2011).
Figure8.MTV9ispredictedtocontaincoiled-coildomains.Predictedcoiled-coildomainsinMTV9aremarkedasgreen
rectangles.
Coiled-coil domains are present in homodimeric membrane tethering factors (Chia and
Gleeson, 2014), involved in intracellular trafficking throughout eukaryotes. To analyze whether
MTV9 could also homodimerize, we tested for interaction by the yeast two-hybrid assay. This
assayshowedthatMTV9caninteractwithitself(Figure9)andmaythushomodimerizeinvivo.
Figure 9. MTV9 interacts with itself in yeast two hybrid assays. pGKB7-MTV9 was cotransformed into AH109
S.cerevisaestrainwithpGADemptyvectorandpGAD-MTV9.Thecontransformantswereplatedonsyntheticselection
mediatoanalyzetheproteininteractionanditsstrength.
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4.MTV9expressioninRNA-seqdatasets
TheATH1microarrayplatformcommonlyusedfortranscriptomicanalysisinArabidopsisdoesnot
containaprobe forMTV9,butwecouldanalyze itsexpression inpublicRNA-seqdatasetsusing
the GENEVESTIGATOR server. MTV9 is a medium to highly expressed gene in most tissues.
Interestingly, gene co-expression analysis showed thatMTV9 is highly co-expressedwith genes
involved or potentially related with intracellular trafficking: among the 25 genes most co-
expressed withMTV9 we found VCL1/VPS16 (Rojo et al., 2001), VPS39, the golgin GC5, ALIX
(Cardona-Lopez et al., 2015), Dynamin2B, and four proteins with phosphatidylinositol binding
domains.
5.MTV9promoteractivity
To study theMTV9 expression profile with cellular resolution, we studied the activity of two
promoter constructs (500 bp or 1200 bp fragments upstream from the start codon) driving
expression of the GUS reporter gene (Figure 10). We analyzed several independent transgenic
linesforeachpromoterconstructandtheyshowedsimilarGUSexpressionpatterns.GUSstaining
wasfirstdetectableinfloraltissues,suggestingthatMTV9expressionismaximalinthosetissues,
inparticularinstamensandinthepetalabscissionzone.Inyoungseedlings,weobservedstrong
GUS expression in the hypocotyl. Transversal cross section of the stem showed that MTV9 is
expressedintheepidermis,thecortexandthevascularbundles(Figure10D).Inrootsandmature
leaves,MTV9expressionisalsodetectableinthevasculatureandin leaftrichomes(Fig.7G),but
not inroothairs.Wealsoobservedexpression in theshootapicalmeristem,which isconsistent
withthemutationaffectingtraffickingofVAC2thereandresultinginmeristemtermination(Figure
10I). In contrast, the expression in root apicalmeristemswas very low. Intriguingly,MTV9was
57
expressed specifically in the quiescent center (QC) (Figure 10L), suggesting that those cellsmay
have special requirements for this gene. We also investigated if treatments with hormones
(auxins, cytokinins, brassinosteroids, gibberellins and jasmonates) or with specific inhibitors of
trafficking (wortmanninandBFA)wouldalterMTV9expression.However,wecouldnotobserve
anysignificantchangescomparedtomocktreatedplants.
Figure10.TissueexpressionoftheProMTV9:GUStransgene.(A)flowermeristem.(B)flowertissue.(C)Gynoeciumwith
pollengrainsonitspistil. (D)Transversalcutofthestem.(E)10days-oldseedlingshowingMTV9expressionmainly in
thehypocotyl.(F)CotyledonshowingMTV9expressioninthevasculartissue.(G)TrichomesinadultleafexpressMTV9.
(H) Root-hypocotyl junction showing expression at the hypocotyl and the vascular tissue of the root. (I) Shoot apical
meristem ina10days-oldseedling. (J)RootshowingMTV9expression in thevascular tissue. (K)15days-oldseedling
showingexpressionintheolderpartsoftheroot.(L)MTV9expressionatthequiescentcenter(QC,arrowhead).
58
6.MTV9localizesprimarilyatthePVC
MTV9may be recruited to the one of the compartments of the vacuolar pathway to exert its
function in vesicular trafficking. Todetermine the subcellular localizationofMTV9,weanalyzed
various versions of the protein fused to theGFP.We cloned theMTV9 coding sequence in the
pGWB5andpGWB6vectorstoproduceinframefusionswithGFPsequenceattheN-terminusand
the C-terminus ofMTV9 expressed under the control of the constitutive 35S promoter (Figure
11A).TransientexpressioninNicotianabenthamianaleavesshowedthatbothversionslocalizedin
a punctate pattern throughout the cytosol. Moreover, transfection of Arabidopsis protoplasts
rendereda similar localizationpattern (Figure11B). These findings suggest that, firstly,MTV9 is
associated with an endomembrane compartment, and secondly, that the localization is not
affectedbyatranslationalfusiontotheGFP,asbothN-terminalandC-terminalversionsshowed
thesameapparentdistribution.
59
Figure11.MTV9showsapunctatepatternintransientlytransformedArabidopsisprotoplastsandNicotianacells.(A)
Nicotiana benthamiana pavement cells agroinfiltrated with 35S-GFP-MTV9 (left) and 35S:MTV9-GFP (right). (B)
Arabidopsisleafcellprotoplaststransfectedwith35S:MTV9-YFP(left)and35S:GFP-MTV9(right).Scalebars:10µm.
Todeterminewhatcompartment(s)correspondedto theobservedpunctatepattern,we
studiedtheco-localizationofGFP-MTV9withmarkersofdifferentendomembranecompartments,
incollaborationwiththegroupofDr.LiwenJiangfromtheChineseUniversityofHongKong.We
co-transformed well-established markers developed in Dr. Jiang’s lab with GFP-MTV9 in
Arabidopsisprotoplasts.Weobservedcompleteco-localizationofGFP-MTV9withthePVC-marker
VSR2-RFP (Figure 12A), indicating that MTV9 is localized at steady state primarily there. In
protoplastswithmoderateexpressionofGFP-MTV9,theGFPsignalwascompletelyseparatefrom
the punctate signal observed for the TGNmarkers SYP41-CFP, and theGolgimarkersManI-RFP
(Figure12Aand13),consistentwithMTV9beinglocalizedinthePVC.Interestingly,weobserved
that in protoplasts overexpressing GFP-MTV9, the RFP-VSR2 signal concentrated in larger dots
(Figure 12B). In order to discard possible effects of the GFP fusion and to confirm that MTV9
overexpression is responsible for thisapparentPVCenlargement,MTV9 fused toa smallHA-tag
was co-transformedwith RFP-VSR2. This experiment also showed accumulation of RFP-VSR2 in
enlargeddots,confirmingthattheeffectoftheMTV9overexpression is independentoftheGFP
fusionattheN-terminus.
60
-
Figure12.MTV9localizesatthePVC.(A)Protoplastswereco-transformedwithGFP-MTV9andRFP-VSR2(PVCmarker),
ManI-RFP(Golgimarker)orSYP41-CFP(TGNmarker).(B)AprotoplastshowinghighlevelsofGFP-MTV9expressionand
PVCaggregation.Scalebars,10µm.
To complement the data from the transient expression systems, we generated stably
transformedArabidopsis plants expressing35S-GFP-MTV9 (pGWB6plasmid). In these lines,GFP
61
fluorescencewasdistributed in a punctate pattern, similar towhatweobserved in protoplasts.
Many of the lines showed transgene silencing and unfortunately, lines showing high expression
were not identified, so the effect of overexpression could not be assessed. For subcellular
localizationstudieswecrossedinmarkersfordifferentcompartments(Geldneretal.,2009).The
PVC markers RHA1-RFP (wave7 line) and ARA7-RFP (wave2 line) co-localized largely with GFP-
MTV9 (Figure 13B,C).Moreover, no co-localizationwas obviouswith the TGNmarker Vha1-RFP
(Figure13A).
62
Figure13.MTV9overexpressionaggregatesthePVCinArabidopsisstabletransformedplants.(A)F1plantsexpressing
GFP-MTV9 and the TGNmarker Vha1-RFP. (B) F1 plants expressingGFP-MTV9 and the PVCmarker Rha1-RFP. (C) F1
plantsexpressingPVCmarkersARA7-YFPandRHA1-RFP.(D),F1plantsexpressingGFP-MTV9andthePVCmarkerARA7-
RFP.Scalebarscorrespondto2,5µmand10µminthecaseofDpanel.
63
Together, theresults fromtransientexpression inArabidopsisprotoplastsor fromstable
expressionintransgenicplantsarecoincidentandsupportthatMTV9associateswiththePVC.
7.TheconservedC-terminaldomainofMTV9isresponsiblefortargetingtothePVC
Analysis of theMTV9 primary sequence in the TMHMM and TMPRED servers for prediction of
transmembranedomainsandintheSignalP4.1Serverforpredictionofsignalpeptides(Petersen
etal.,2011) suggest thatMTV9proteindoesnotcontaina signalpeptidenora transmembrane
domainforassociationwithmembranes.To identifythesequencesresponsible forthetargeting
to the PVCwedissected theMTV9 coding sequence in three non-overlapping constructs, fused
them toGFP (Figure14A) andanalyzed their localization inN.benthamiana leaves (Figure14B)
andinArabidopsisprotoplasts(Figure15).ThefragmentsanalyzedwereNT(aminoacids1-199),
CORE(aminoacids200-472)andCT(aminoacids473-678).ThelocalizationofNT-GFP,CORE-GFP
andNT-CORE-GFPwascytosolic,andnoassociationwithmembranecompartmentswasapparent.
Incontrast,theCORE-CT-GFPandtheCT-GFPconstructspresentedapunctatepatternsimilarto
the full length MTV9 (Figure 14B). Similar results were obtained in Arabidopsis protoplasts
transformedwiththeseconstructs(Figure15).TheseresultssuggestthattheCTfragmentcontains
thenecessary information for targetingMTV9 to the PVC. TheCT is themost conserved region
among MTV9 homologues and the STV9 paralogue, which also localizes to endosomes (Figure
14C),indicatingthattheCTisinvolvedinmembranerecruitmentandthatthisisessentialforthe
functionoftheseproteins.
64
Figure 14. The CT domain is necessary for correct localization ofMTV9. (A), different deletion constructs ofMTV9
clonedintothepGWB6vector.(B),GFP-localizationofthedifferentdeletionformsofMTV9inN.benthamianaleaves.
(C), i) STV9 construct that carriesRFP fused to STV9 cDNAunder the control of the constitutivepromoterUBQ10, ii)
localizationofRFP-STV9inN.benthamianaleafpavementcells.Scalebarscorrespondto10µmand2,5µminthecase
ofSTV9picturemagnification.
65
Figure15.TheCTdomaindirectslocalizationatthePVC.(A),N-terminalpartofMTV9co-transformedwithRFP-VSR2.
(B), N-terminal part and CORE domain of MTV9 co-transformed with RFP-VSR2. (C), CORE domain of MTV9 co-
transformed with RFP-VSR2. (D), CORE domain and C-terminal part of MTV9 co-transformed with RFP-VSR2. (E), C-
terminalpartofMTV9co-transformedwithRFP-VSR2.Scalebars,10µm.
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8.MTV9overexpressionperturbstransportofvacuolarcargobutnotofPMproteins
Asnotedabove,MTV9overexpressioninprotoplastscausedtheRFP-VSR2signaltoconcentratein
largerdots,whereasmoderateexpressiondidnot cause this apparentPVCenlargement (Figure
12). Co-transformation experiments with the TGN syntaxin markers RFP-SYP61 and SYP41-CFP
showed that in protoplast expressingmoderate amounts ofGFP-MTV9, theGFP-MTV9 signal at
the PVC was separated from RFP-SYP61 and SYP41-CFP signal at the TGN (Figure 16A).
Interestinglyhowever,whenGFP-MTV9wasstronglyoverexpressed,SYP61butnotSYP41wasre-
localizedintheenlargedGFP-MTV9positivecompartments(Figure16B).
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-
Figure 16.MTV9overexpression leads to SYP61mislocalization. (A). Protoplasts expressingmoderate levels ofGFP-
MTV9co-transformedwithSYP41-CFPorRFP-SYP61.(B),ProtoplastsexpressinghighlevelsofGFP-MTV9co-transformed
withSYP41-CFPorRFP-SYP61.Scalebars,10µm.
We reasoned that PVC enlargement and SNARE mislocalization caused by MTV9
overexpression should interfere with the functionality of the compartment and with protein
traffickingtothevacuole.Totestthis,weco-transformedArabidopsisprotoplastswithGFP-MTV9
68
andasolublevacuolarcargo(Aleurain-RFP),atonoplastmembranevacuolarcargo(RFP-VIT1)and
aplasmamembranecargo(RFP-SCAMP1).AleurainisathiolproteasewithanN-terminalvacuolar
sorting signal that has beenwidely used as amodel soluble vacuolar cargo.When transformed
into Arabidopsis protoplast, it labels the lumen of the vacuole.When it is co-transformedwith
GFP-MTV9,Aleurain-RFPalsoreachesthevacuolebutthereisalsosomeretentionintheenlarged
PVCs together with MTV9-GFP (Figure 17), suggesting that trafficking to the vacuole is partly
disrupted.Solubleandmembranecargoesmayusedifferent routes to reach thevacuole, sowe
analyzedhow transport of the tonoplastmembranemarkerwas affectedby co-expressionwith
MTV9-GFP. Interestingly, RFP-VIT1 was mostly found in the enlarged PVCs together with GFP-
MTV9(Figure17)andalmostnoneoftheproteinreachedthetonoplast.Hence,theinterference
with targeting of thismembrane cargowas higher thanwith aleurain, possibly because soluble
cargocanentervesiclesfromalternativevacuolartransportpathways.Totestiftheeffectswere
specific to vacuolar proteins, we co-transformed the PMmarker RFP-SCAMP1 withMTV9-GFP.
Importantly,SCAMP1-RFPwasproperlytargetedtothePM,indicatingthatonlytraffickingtothe
vacuolewasdisrupted.ThisisconsistentwithMTV9overexpressionaffectingthefunctionalityof
thePVC,alateendosomespecificforthevacuolartransportroute.
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Figure17.TraffickingtothevacuoleisdisruptedwhenMTV9isoverexpressed.ArabidopsisprotoplastsexpressingGFP-
MTV9andthevacuolarsolublecargoAleurain-RFP,theplasmamembraneproteinRFP-SCAMPorthetonoplastprotein
RFP-VIT1.Scalebars,10µm.
9.PVCaggregationbyMTV9overexpressionisnotaffectedbywortmannin
Wortmannin,aPI3kinaseinhibitor,causeshomotypicfusionofPVCsleadingtotheformationof
greatlyenlargedPVCsthatappearassphericalcompartmentswithalimitingmembranethatcan
beresolvedfromthelumeninalightmicroscope(Figure18A).Thisisdifferentfromtheenlarged
PVCsformedinMTV9-GFPoverexpressingprotoplasts,whichhaveamoreamorphousshapeand
thelumenisnotdistinguished.ThissuggeststhatthemechanismcausingPVCenlargementinthe
MTV9-GFP overexpressing protoplasts is different from that of wortmannin and may instead
reflect an aggregation of non-fusing PVCs. This process of aggregation of intracellular
70
compartments in certain cytosolic domains is also observed in plants treated with BFA, which
induces the formation of the so-called BFA bodies that are large aggregates of Golgi and TGN
cisternaeinthecytosol.GiventhatwortmanninandMTV9overexpressionwerebothaffectingthe
PVC,weanalyzedtheeffectoftheircombinedaction.Interestingly,wortmannintreatmenthadno
effectonPVCmorphologyinprotoplasts(Figure18A)orintransgeniclinesoverexpressingMTV9-
GFP(Figure19),indicatingthattheMTV9-inducedaggregationofthePVCpreventedwortmannin-
induced homotypic fusion. To confirm this, we co-transformed protoplast with a constitutively
active formof ARA7(Q69L) that also causes homotypic fusion of PVCs andmimics the effect of
wortmannin treatment (Jia et al., 2013). Protoplasts transformedwith ARA7(Q69L)-RFP induced
the formationof large ring–likePVCs,but thiseffectwas lostwhenco-transformedwithMTV9-
GFP,whichledtoPVCaggregation(Figure18B).
Figure 18. PVC aggregates caused byGFP-MTV9 overexpression are not affected bywortmannin treatments or by
constitutivelyactiveARA7.(A)Arabidopsisprotoplastsco-transformedwithGFP-MTV9andRFP-VSR2(leftpanels)and
treatedwithwortmannin do not produce ring-like structures observed in RFP-VSR2 transformed protoplasts treated
with wortmannin (right panels). (B) GFP-MTV9 overexpression changes the ring-like structures of ARA7 (Q69L)
71
transformedprotoplasts(leftpanels),andaggregatesthePVCinARA7-RFPtransformedprotoplasts(rightpanels).Scale
bars,10µm.
Together, these experiments suggest thatMTV9 overexpression causes a rearrangement of the
PVCsintoaggregates,whichimpedesnormalfunctionalityofthePVCandblockswortmannin-and
ARA7(Q69L)-inducedhomotypicfusionofthecompartment.
Figure19.Wortmannindidnotproduceenlargedring-likePVCsinMTV9oeplants. InArabidopsisplantstransformed
withARA7-YFPthePVCsshowthetypicalringshape(arrowhead)aftertreatmentwith33µMwortmannintreatment.In
contrast, in plants expressing GFP-MTV9 the aggregated PVCs do not respond to wortmannin treatment. Scale bars
correspondto10µm(control:mocktreatedplants)and2,5µm(Wm:wortmannintreatedplants).
10.CharacterizationofantibodiesagainstMTV9
To characterize the endogenousMTV9protein,we raisedpolyclonal antibodies against the first
472aminoacids (NT-CORE fragment)ofMTV9.Wecloned the fragment in thepDEST17plasmid
andexpresseditinEscherichiacoli.Figure20showsthatwithinthreehoursofinductiontherewas
highexpressionoftherecombinantproteinthataccumulatedalmostentirelyininclusionbodies.
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Figure20.RecombinantexpressionoftheNT-COREMTV9fragment(aminoacids1-472)inE.coli.Proteinextractsof
theE.coliculturesinducedwithIPTGfor0,1.5and3hwereanalyzedbySDS-pageandcoomasiestaining.i:insoluble
fraction;s:solublefraction.
We purified inclusion bodies containing high concentration of the recombinant protein
andusedthemtoimmunizerabbits.Theobtainedserashowedaspecificrecognitionofaprotein
band of the expected size (90 kDa) in samples fromWild-type (Wt) plants that was absent in
extracts from mtv9-2 mutant plants (Figure 21), indicating that it corresponds to the MTV9
protein.
Figure21.TheMTV9antibodyspecifically recognizesaproteinbandabsent inmtv9-2proteinextracts.Theasterisk
indicatestheproteinbandspecificallyrecognizedbytheMTV9antibodyinstemandflowerproteinextracts.
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11.MTV9associateswithmembranesthatcorrespondtotheTGNandthePVC
Togain insight intothe localizationof theendogenousMTV9protein,wecarriedoutsubcellular
fractionation studies using the antibody for detection. First we performed differential
centrifugationexperimentswith samples fromWt seedlings.As shown in Figure22A, theMTV9
protein was found in the pellet fractions, consistent with association with endomembrane
compartments.ThefractionationpatternwasverysimilartothatofSYP21,aQa-SNAREresidingat
the PVC. To determine how MTV9 associates with membranes, we isolated microsomal
membranes and tested for extractionwithdifferent agents, using SYP21, an integralmembrane
protein, and VPS45, a peripheral membrane protein, as controls. Interestingly, MTV9 behaved
similarly toSYP21andcouldonlybeefficientlyextracted frommembraneswithTritonX-100. In
contrast,VPS45wasalsoextracted in thepresenceofurea. These results indicate thatMTV9 is
likelyinsertedintothemembrane(Figure22B).However,MTV9doesnotcontaintransmembrane
domains, so the insertion should be through post-translationalmodificationwith lipid anchors..
Interestingly,ithasbeenrecentlyreportedthatMTV9maybepalmitoylated(Hemsleyetal.,2013)
whichcouldservetoattachtheproteintomembranes.Palmitoylationnormallyoccursatcysteine
residues andMTV9 contains a single cysteine that is present in the CT domain, and may thus
promotePVCtargetingoftheprotein.
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Figure22.MTV9istightlyassociatedwithmembranes(A)Differentialcentrifugationofproteinsamplesfrom10days-
oldCol-0ormtv9-2seedlings.Thenumbersindicatecentrifugalforceappliedtotheproteinsample(inthousandsg),the
“S”indicatessoluble,and“P”indicatesthepelletfraction.(B)100,000gmembranepelletfromanArabidopsis10days-
oldseedlingsextractwasresuspendedinHEbufferalone,orin2Murea,1MNaCl,0.1MNa2CO3,or1%TritonX-100,
respectively.Insolublematerialwasre-pelleted,andsolubleandpelletfractionswereanalyzedbySDS-PAGEwithanti-
MTV9, anti-SYP21 (integral membrane protein localized to the PVC) and anti-VPS45 (peripheral membrane protein
localizedtotheTGN).
In sucrose gradients, themajority ofMTV9was found in fractions containing the PVC resident
proteins VTI11 and SYP21, while VSR proteins that cycle between the TGN and the PVC only
partiallyoverlappedwithMTV9(Figure23).ThesubcellularfractionationpatternofMTV9(Figures
22and23)isconsistentwiththelocalizationoftheGFPfusionconstructsinthePVCandsupports
thattheendogenousMTV9localizestothatcompartment.
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Figure 23. MTV9 associates with membrane fractions that correspond to the prevacuolar compartment. Sucrose
gradientoftendaysoldsseedlingsfromCol-0wereanalyzedbywesternblotswithantibodiesagainstMTV9andVTI11,
VSR,andSYP21,thepreviouslycharacterizedorganellemarkers.
12.MTV9andVTI11functioninseparatevacuolartraffickingpathways
Noneof the four isolatedmtv9mutant allelespresentedobviousphenotypes in the absenceof
VAC2,exceptforaslightbutsignificantdelay infloweringtime(Figure24).Totest if this lackof
phenotypewasduetothegeneredundancyoftheSTV9gene,weisolatedandcharacterizedtwo
T-DNAinsertionsinthatgene.Thestv9mutantallelesdidnotcausesecretionofVAC2whenthe
transgenewascrossedin,arguingagainstSTV9havingthesamefunctionsasMTV9.Moreover,an
mtv9-2stv9-1doublemutant,whichispredictedtocarrynullallelesinbothgenes,didnotdisplay
anyotherdevelopmentalphenotypethanthedelayedfloweringalreadyobservedinsinglemtv9-2
mutants.Hence,itappearsthatMTV9andSTV9arenotessentialforplantviabilityorfornormal
development.
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Figure24.mtv9-2floweringtimeisslightlydelayedunderlongdayconditions.(A)4weekoldplantsfrommtv9-2and
Col-0backgroundsshowingthemtv9-2floweringdelay(B)Theaveragenumberofrosetteleavesorrosettepluscauline
leavesatthetimeofpetalemergenceinthefirstflower(54plantspergenotype).Theerrorbarsrepresentthestandard
deviation.AnunpairedStudent`st-testwasusedtocomparethemeans.Twoasterisks:p-value<0.001;threeasterisks:
p-value<0.0001.
The lack of any obvious phenotype in the mtv9-2 stv9-1 double mutants is striking
considering the conservation of theMTV9 gene family in plants and the drastic effect ofMTV9
overexpressiononPVCmorphologyandfunctionality.Apossibleexplanationwouldbethatthere
are MTV9-independent pathways to the vacuole that mask the effects of blocking the MTV9-
dependent pathway. To gain genetic evidence for this, we crossed mtv9 with previously
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characterizedvacuolar traffickingmutants (mtv1,mtv2/vsr4, vsr1,mtv3,mtv4,mtv11, vti12 and
vti11), with the rationale that alterations in parallel pathways should have a synergistic effect
whencombinedwiththemtv9mutations.Thedoublemutantsfromallthecrossesdidnotshow
any increased phenotypic changes in standard growth conditions relative to the respective
parental single mutants, except for themtv9 vti11 double mutant that showed more severe
dwarfismthanthesinglevti11mutant(Figure25and26).
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Figure 25. Growth phenotypes of single and double mutant combinations. Only the vti11mtv9 mutant shows
synergisticeffectsongrowthinhibitionrelativetothesinglemutantparents.
Figure26.mtv9enhancesthephenotypeofvti11mutant.Thepicturescorrespondto6weekoldplantsgrownunder
longdayconditions.
Analysisofseedstorageprecursoraccumulationinthedoublemutantsgenerateddidnot
reveal defects in processing in any of themutants (Figure 27), indicating that at least in seeds,
vacuolartraffickingwasstillproceedingefficiently.Incontrast,inleaveswecoulddetectabnormal
secretionofavacuolarperoxidaseinthemtv9-2vti11mutant,consistentwithvacuolartrafficking
beingsynergisticallyperturbedinvegetativetissuesofthedoublemutant.Consideringthatmtv9-
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2 and vti11 are likely null alleles, these results suggest that MTV9 and VTI11 function in
independentvacuolartraffickingpathways.
Figure27.mtv9doesnotalterthetraffickingofstorageproteinsinseeds.ThelanescontainCol-0seedproteinextract
asacontrolandseedextractsfrommtv9-2mutantandindicateddoublemutantsofmtv9-2.
13.MTV11encodesahomologueofyeastVPS15.
Themtv11-1mutantwasisolatedfrompool32.TheF1plantsfromacrossbetweenmtv11-1and
Col-0 had a wild type phenotype, and in the F2 population, the proportion of plants withmtv
phenotypewasconsistentwitharecessivemutation.Weperformedbulk-segregantanalysiswith
DNAfromapoolof50F2mtvplantsandfoundaregionofchromosomeIVshowingenrichmentin
Ler SSLPs. Fine mapping restricted the interval containing themtv11-1mutation to a 0.26Mb
region(between14.40and14.66Mb)(Figure28).
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Figure28.mtv11mappedtoa0.26MbregiononchromosomeIV.Thenumberofrecombinantplantsforthedifferent
molecularmarkers is indicated. 5 recombinant plants delimited a region between 14.4 and 14.66Mb containing the
mtv11mutation.
TheregiondelimitedthroughmappingcontainedtheAt4g29380genethatencodesahomologue
ofVPS15, anessentialgene forvacuolar traffickingandautophagy inyeastandanimals (Anding
andBaehrecke,2015;HermanandEmr,1990;Lindmoetal.,2008).SequencingofAt4g29380 in
mtv11-1plantsrevealedanEMS(G/A)mutationthatdisruptedthespliceacceptorsiteofthe7th
intron(Figure29C).
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Figure29.mtv11encodesVPS15protein.A)DomainspresentinMTV11/AtVPS15B)Terminatedshootmeristem
phenotypeofthemtv11-1mutant,C)SchematicrepresentationoftheMTV11/AtVPS15genelocus,showingthemtv11-
1EMSmutationatthespliceacceptorsiteofthe7thintronofAtVPS15andthemtv11-2T-DNAinsertion.
Analysis by RT-PCR revealed that most of the At4g29380/AtVPS15 transcripts in the
mtv11-1 plants retained the 7th intron (Figure 30), which introduces an early stop codon that
deletes most of the coding sequence of the protein. Moreover, we also detected a significant
amount of mRNAmolecules in the mutant with the 7th intron spliced out using an alternative
spliceacceptorsite4nucleotidesdownstreamoftheoriginalone,whichcausesaframeshiftand
also leads toanearly stopcodon.These twosplicevariants (7th intronretentionandalternative
spliceacceptorsite inexon8)wouldproduceatruncatedMTV11/VPS15proteincontainingonly
thekinasedomain.Interestingly,wealsoobservedinmtv11-1skippingoftheeighthexonbyusing
anintronacceptorsiteinthe8thintron.Thisexonskippingeventmaintainsthereadingframeand
the resultant transcript would produce a truncated protein containing the kinase domain and
threeofthefourWD.
A
B
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Figure30.Themtv11-1mutantaccumulatesdifferentially spliced transcripts. (A)RT-PCRanalysisof the retentionof
the7thintronofMTV11inWtandmtv11-1mutantplantswithflankingprimers.(B)SchematicrepresentationoftheWt
MTV11/AtVPS15protein and the two truncated versions encodedby thedifferentially spliced transcripts detected in
mtv11-1plants.
Intriguingly, null mutations in AtVPS15 show pollen lethality and thus homozygous
mutants cannot be recovered (Wang et al., 2012; Xu et al., 2011), indicating that themtv11-1
alleleretainspartialactivityofthegene.ToconfirmthatthemutationinAt4g29380/AtVPS15was
the cause of the mtv phenotype, we crossed a heterozygous T-DNA null mutant (atvps15-
2/Salk_004719)withmtv11-1homozygousforVAC2.IntheF1,weobservedplantseitherwithWt
orwithmtvphenotype.GenotypingusingaCAPSmarkerformtv11-1andflankingprimersforthe
T-DNA insertion showed that plants withmtv phenotype corresponded tomtv11-1/atvps15-2
plants andplantswithWtphenotype tomtv11-1/MTV11 plants.Hence,mtv11-1 andatvps15-2
areallelic,whichconfirmsthattheactivityofMTV11/AtVPS15isnecessaryfortransportofVAC2
tothevacuole.
Toanalyze thephenotypeofmtv11-1plants in theabsenceofVAC2orother secondary
EMSmutations,webackcrossedtheplant5timeswiththeparentalLergenotypeandsegregated
out theVAC2 transgene. Themtv11-1 showed a reduced growth rate in soil (Figure 31) and an
altered phyllotaxis, possibly due to alterations in vacuolar turnover of PIN auxin-efflux
transporters.
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Figure31.GrowthphenotypeofWt(Ler)andmtv11-1plants.
We analyzed if transport of endogenous seed storage proteinswas also affected in the
mutant. As shown in Figure 32, themtv11-1mutant accumulated precursors of 12S globulins,
indicatingthattransportofthesestorageproteinstothevacuolewashindered(Fig32A).Protein
storage vacuoles (PSV) in seeds are autofluorescent and can thus be visualized by fluorescence
microscopy. Interestingly, PSVs inmtv11-1 embryos had lower fluorescence levels than in Wt
embryos(Figure32B),consistentwithlowerlevelsofproteinaccumulation.Weconcludeforthese
analyses that MTV11/AtVPS15 is required for vacuolar trafficking of both exogenous and
endogenousvacuolarcargo.
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Figure32.MTV11 is involved in transportof 12S globulins. (A) Total seedprotein extracts showing accumulationof
precursor form of 12S globulin (p12S) inmtv11 (left panel). (B) Autofluorescence of protein storage vacuoles in
hypocotylcellsfrommatureWtandmtv11-1embryos.Scalebar10µm.
14.MTV11/AtVPS15localizestoendosomalcompartments
To study the localization ofMTV11/AtVPS15,we fusedGFP at theN andC-terminal end of the
proteinandexpressedtheseconstructsinNicotianabenthamianaleaves.GFP-AtVPS15showeda
cytosolic localizationwhereasAtVPS15-GFP showedboth cytosolic andpunctate pattern (Figure
33), suggesting that there are soluble and membrane bound pools of the fusion protein.
Interestingly,MTV11 has an N-terminal glycine residue that is conserved in VPS15 homologues
from plants, animals and fungi. Moreover, it has been reported that the N-terminal glycine is
myristoylated in yeast VPS15 (Herman and Emr, 1990). This myristoylation motif would be
maintained in theAtVPS15-GFPbutnot inGFP-AtVPS15, explaining thedifference in subcellular
localizationandsuggestingthatadditionofmyristicacidisnecessaryforreversibleattachmentof
MTV11 to membranes. When expressed in Arabidopsis protoplasts, GFP-AtVPS15 was also
cytosolic. Inprotoplasts,AtVPS15-GFPshowedmainlya cytosolicpatternandonly ina fewcells
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some punctate structures could be observed. Stable 35S-AtVPS15-GFP transgenic lines were
generated but they had very low levels of transgene expression and could not be used for co-
localizationexperiments.Hence,wehaveyet to identify the compartmentswhereAtVPS15-GFP
localizes.
Figure33. Subcellular localizationofGFP-taggedMTV11/AtVPS15 in tobacco leaf cells.MTV11/AtVPS15was tagged
with GFP at the N-teminal (GFP-AtVPS15) or C-terminal (AtVPS15-GFP) of the protein and expressed in Nicotiana
benthamianaleafepidermiscells.Scalebar=100µm.
15.Themtv11-1mutanthasreducedPI3Plevels
VPS15 is a subunit of phosphatidylinositol 3-kinase from class III (PIK3C3) that is essential for
membranerecruitmentofthecomplexandforactivationofthecatalyticVPS34subunit(Stackand
Emr,1994;Stacketal.,1993).TheactivatedPIK3C3phosphorylatesphosphatidylinositol(PI)atD-3
positions to form PI3P. The localized synthesis of PI3P functions as a signal for membrane
recruitment of effector proteins involved in vesicle trafficking and autophagy. The specificity of
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ArabidopsisthalianaPIK3C3isstillunknown.TodetermineifMTV11isinvolvedinregulatingPI3P
levels in vivo, wemade use of the PI3P biosensor line YFP-2xFYVE, consisting on YFP fused to
tandemdimeroftheFYVEdomainofmouseHsr,whichspecificallybindsPI3P,underthecontrol
of the constitutive35Spromoter (Vermeeret al., 2006).Wecrossed transgenic linesexpressing
2xFYVE (inCol-0background)withmtv11-1mutantsbackcrossed4 times intoCol-0background
andobtained in theF2mtv11-1plantshomozygous for2xFYVE.Comparisonof the fluorescence
pattern between these plants and the parental YFP-2xFYVE line showed clearly reduced
fluorescence in the mtv11-1 mutants. Whereas in Wt plants, YFP-2xFYVE strongly labelled
numerous endosomes, in many cases forming large aggregates, in mtv11-1 plants overall
fluorescencewaslowerandthe2xFYVElabeledendosomesremainedmostlyisolated(Figure34).
TheseresultssupportthatMTV11/AtVPS15functionsasanactivatoroftheVPS34PI3Pkinasein
plants.Consistentwiththis,AtVPS15interactswithAtVPS34inyeasttwohybridassays(Wanget
al., 2012) and defects in pollen germination of vps15 mutants are rescued with external
applicationof PI3P (Xu et al., 2011). The reduction in PI3P levels in endosomeswould interfere
with recruitment of trafficking effectors, explaining why vacuolar transport is perturbed in the
mtv11-1mutant.
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Figure 34. Altered distribution and reduced levels of PI3P in endosomes ofmtv11-1 revealed by the YFP-2xFYVE
biosensor.Scalebar25µm.
16.Themtv11-1mutantsshowincreasedgrowthinarsenatecontainingmedia.
Manytoxiccompoundsaresequesteredintothevacuolesfordetoxifixation.Wethuswonderedif
defective vacuolar trafficking in mtv9 and mtv11 mutants would affect vacuolar-mediated
detoxificationcapacity.Totest this,we focusedonarsenic,oneof themost toxiccompounds in
soil.Arsenicisfoundinsoilsmainlyasarsenate[As(V)]thatistransportedintocellsbyphosphate
transporterspresent in thePM. In the cytosol arsenate is rapidly reduced into arsenite [As(III)],
complexedtosolublethiols,andtransportedintothevacuolefordetoxification(Catarechaetal.,
2007; Sanchez-Bermejoet al., 2014). Todetermine if arsenic tolerancewasaltered inmtv9 and
mtv11mutants,wemeasuredincollaborationwiththegroupofDr.AntonioLeyvaattheCNBthe
root growth dynamics of Wt and mutant plants under different concentrations of As(V). We
comparedmtv9-2withitsWtbackground(Col-0)andmtv11-1(backcrossed5timesintoLer)with
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itsWtbackground(Ler).Wedidnot findanydifferences inAs(V)-inducedrootgrowth inhibition
betweenWtandmtv9-2plants,but strongdifferenceswereobservedbetweenmtv11-1 and its
WtcontrolinmediaccontaininghighAs(V)concentration(Figure35).Themtv11-1plantsshowed
significantlowerlevelsofrootgrowthinhibition,indicatingthattheyhaveincreasedtoleranceto
As(V)eventhoughtraffickingtovacuolesisdisturbed.
Figure35.mtv11-1showsenhancedgrowthinarseniccontainingmedia.Rootgrowthofthegenotypesindicatedinthe
graphswasmeasured inmediawith lowphosphate(-Pi:12,5µMphosphate)or inthesamemediawith10or20µM
As(V).Theaveragegrowth(relativetogrowthoftheWtin–Pimedia)isshown.Errorbars:standarddeviation.Asterisks:
significantdifferencerelativetotheWtinthatmedia(p<0.001;Student´st-test).
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Apossibleexplanationtothissurprisingresult,consideringthatperturbationstovacuoles
were expected to interfere with detoxification, is that MTV11 participates in other trafficking
routes that require PI3P andwould be necessary for As(V) uptake into the plant. For instance,
enhanced tolerance would be expected if the mtv11-1 mutation interfered with targeting of
phosphatetransporterstothePM.Totestthis,wecrossedthemtv11-1mutantwithamarkerline
expressingthehighaffinityphosphatetransporterPHT1;1fusedtoGFPunderthecontrolofthe
35Spromoter(Gonzalezetal.,2005).Withlowphosphateinthemedia(-Pi:12,5µMphosphate),
PHT1;1-GFP was found primarily in the PM, both in Wt plants and inmtv11-1 mutants. The
presenceof30µMAs(V)in-PimediainducedturnoverofPHT1;1-GFPfromthePMinWtplants
(Figure36)aspreviouslyreported(Castrilloetal.,2013).Interestingly,themtv11-1plantsshowed
higherlevelsofPHT1;1-GFPinthePMafterexposuretoAs(V)andinadditiontheyshowedstrong
accumulationofPHT1;1-GFPinendocyticcompartments.Similarly,inphosphaterichmedia(+Pi:1
mMphosphate),mtv11-1plantsshowedhigherlevelsofPHT1;1-GFPinthePMthanWtplantsand
also significant accumulation in endocytic compartments. The detection of PHT1;1-GFP in
endocytic compartments is likely due to the perturbations in vacuolar trafficking, which would
delay targeting of the protein to the vacuole for degradation (Cardona-Lopez et al., 2015).
Importantly, these results indicate that the higher As(V) tolerance ofmtv11-1 plants cannot be
ascribedto lower levelsofPHTtransporters inthePM,butmay insteadbeduetoalterations in
the vacuole than somehow favor As(III) uptake into that compartment. A possibility is that the
concentration of arsenite conjugate transporters in the tonoplast is relatively increased in the
mutantorthatlowerlevelsofproteinaccumulationpermithigherarseniteuptake.
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Figure36.PHT1;1isretainedinendosomalcompartmentsinmtv11-1background.SubcellulardistributionofPHT1;1-
GFPinWtandmtv11-1roots.ThedistributionofPHT1;1-GFPinlowphosphatemedium(-Pi),inhighphosphatemedium
(+Pi)andinlowphosphatemedium24hafteradding30µMAs(V)(-Pi+As(V)24h)isshown.Scalebar2,5µm.
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Discussion
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1.PhenotypicconsequencesofdisruptingMTV9activityMap-basedcloningofthemtv9mutantrevealedthatdefectivetraffickingoftheVAC2proteinwas
due to a point mutation introducing an early stop codon in At1g24560, a gene of unknown
function.HomologuesofMTV9/At1g24560arenotpresentoutsidetheplantkingdomorinalgae.
In contrast, all land plants contain homologues of MTV9, including Physcomitrella patens and
Marchantia polymorpha. This could indicate that MTV9 may have functions related with the
specificneedsoflandplants,suchasresistancetosalinityordrought,asdescribedforARA6(Ebine
et al., 2011). Vacuolar trafficking is an essential cellular process, and disruption of vacuolar
traffickingmachineryoftenleadstoplantlethalityorseveregrowthdefects(Niihamaetal.,2005;
Rojo et al., 2001; Sanmartin et al., 2007; Sauer et al., 2013). The mtv9 mutants have weak
phenotypes, but there is aparalogueofMTV9 inArabidopsis, theSTV9/At3g49055 gene,which
couldbegeneticallyredundantwithMTV9.However,thestv9mutantdoesnothaveterminated
meristems in thepresenceof theVAC2 transgene, indicating that its activity isnotessential for
trafficking of this cargo, possibly due to redundancy fromMTV9. Moreover,mtv9 stv9 double
mutants do not display any additional phenotypes in normal growth conditions relative to the
mtv9singlemutants,supportingthatthegenesdonothaveanessentialfunctionunderstandard
growth conditions.However, it is possible that phenotypesmaybecomeapparent in conditions
requiring optimal vacuolar trafficking capacity or under stress conditions. Moreover, it is also
possible that the alleles analyzed may still retain some gene activity, although this is unlikely
because the T-DNAs are inserted in exons and should eliminatemost of the coding sequences.
AnotherpossibilityisthatthecargotransportedthroughtheMTV9/STV9-dependentpathwaymay
bereroutedtoanMTV9/STV9-independentpathwayandstillreachthevacuole inthemtv9stv9
mutant,whichwouldexplainwhyphenotypesarenotclearlyobservable.Furthermore,MTV9and
STV9maycooperatewithotherproteins toachieveaparticular trafficking reaction (seebelow),
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and strong defectsmay only become evident if both factors are disrupted. An example of this
would be theMTV1 andMTV4 genes, which belong to distinct gene families butmay both be
involved in clathrin coated vesicle formation at the TGN. Only when both genes are mutated,
phenotypicconsequencesatthewholeplantscaleareevident(Saueretal.,2013).
2.FunctionaldomainsinMTV9
AsearchforknowndomainsinthePfamwebsitesuggeststhattheMTV9proteincontainscoiled-
coil domains along its sequence. Coiled-coiled domains may be involved in homo or hetero
oligomerization and may also function as molecular spacers (Truebestein and Leonard, 2016).
Coiled-coildomainsare found inhomodimericmembranetethering factorssuchasgolgins (Chia
andGleeson,2014).MTV9homodimerizesandcouldalsofunctionasamembranetetheringfactor
at the PVC. Indeed, the PVC aggregation caused byMTV9 overexpression supports that itmay
tether incoming vesicles or endosomal compartments for homotypic or heterotypic fusion.
Interestingly,MTV9 is highly co-expressedwith VCL1 and VPS39,which are components of the
CORVET and HOPS multisubunit tethering complexes that regulate homotypic fusion of
endosomes to formthePVCand theheterotypic fusionof thePVCwith thevacuole in the final
step of the vacuolar trafficking pathway (Chia andGleeson, 2014; Rojo et al., 2001; Rojo et al.,
2003).Homodimerizingmembranetetheringfactorsworkatlongranges(ChiaandGleeson,2014),
whilemultisubunittetheringcomplexesfunctionatshorterdistances.ItisthuspossiblethatMTV9
cooperateswiththeCORVETand/ortheHOPScomplexestomediatemembranefusionatthePVC.
Then,anexplanationtothelackofphenotypeofthemtv9mutantscouldbethatMTV9improves
theefficiencyorspeedsuptethering,butisnotessentialfortheprocess.Incontrast,theCORVET
and HOPS complexes appear to be essential for tethering, and knockout mutations abrogate
vacuoleformationandimpairplantviability(Rojoetal.,2001).However,wehaverecentlyisolated
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a novelmtv mutant with a hypomorphic mutation in aVPS39-like gene from the TRAP1 clade
(Klinger et al., 2013). Yeast VPS39 is a subunit of theHOPS complex that is distantly related to
yeastVPS3,asubunitoftheclassCcorevacuole/endosometethering(CORVET)complex(Klinger
etal.,2013).Interestingly,animalandplantgenomescontainseveralclosehomologuesofVPS39,
but not of VPS3. It has been proposed that the human VPS39-like homologue TRAP1 could
substituteforVPS3intheCORVETcomplex(Lachmannetal.,2014,Klingeretal.,2013).Itisthus
possiblethattheArabidopsisVPS39-likegenealsoassumesthefunctionoftheVPS3subunitinthe
CORVET complex, which functions as a Rab5 effector and coordinates membrane fusion at
early/lateendosomes.Knockoutmutations in thisVPS39-likegeneareembryonic lethal,but the
viablehypomorphicallelethatwehave isolatedwillallowustotestwhether indeedit ispartof
the CORVET complex, andwhether this complex cooperates withMTV9 for tethering incoming
vesiclesat thePVC. If thatwere the case,we should find stronggenetic interaction inadouble
mtv9vps39-Lmutant.
The fact that MTV9 and STV9 genes are specific for land plants suggests that these
organisms may have exclusive needs that require the function of these genes. Because MTV9
homologues are present in basal plants like Physcomitrella andMarchantia, it implies that the
plantspecificneedspredatetheappearanceofseedplants.InthatregarditistellingthatMTV9is
not required for vacuolar trafficking of storageproteins in seeds, but it is required for vacuolar
trafficking in vegetative cells. Clearly, vacuoles in seed cells and in vegetative cells are strikingly
different(ZouharandRojo,2009)andsoistheorganizationofintracellularcompartments,sothe
trafficking requirements may be different. Indeed, a key adaptation of land plants was the
acquisitioninvegetativecellsoflargevacuolesthatoccupymostofthecellularvolume,incontrast
toalgal,yeastandmammaliancellsthatcontainsmallvacuoles.Thelargevacuolesinlandplants
allowforenergeticallycheapgrowththat isessential fortheseautotrophicorganismstoexplore
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the surrounding environment for light,water and nutrients. In addition, the large vacuoles also
serve tomaintain cellular homeostasis by acting as a buffering compartment (Zouhar andRojo,
2009). It is evident that having such large vacuoles alters considerably the organization of the
cytosol and the organelles within it, and therefore the trafficking between the different
compartments. It is then foreseeable the need for novel tethering factors that help vesicles to
navigate this new cellular landscape and promote efficient membrane fusion reactions. In this
regard,MTV9 (and STV9)may have been selected to perform an activity essential for effective
vacuolar trafficking in thesenovel cellular settings.However, in standardgrowth conditions,we
havenotdetectedanydeleteriousconsequencesofknockingoutMTV9andSTV9,arguingthatthe
activityofthesegenesislargelyirrelevantfornormalfunctioningoftheplant.Thechallengethen
istofindifthereareconditionsinwhichalteringthistheiractivityhasphenotypicconsequences
fortheplant.Apossibilityisthattheiractivityisespeciallyimportantfortheresponsetostresses
specifically encountered by land plants, such as salt and drought stress, or for processes that
involveactivevacuoledynamics,suchasstomatalmovements.Therefore,itwillthenbeimportant
to test all the doublemutants generated, and especially themtv9stv9 doublemutant, under a
battery of stress conditions to find conditions in which the activity of this gene family has an
essential role. Asmentioned above,Marchantia polymorpha has a singleMTV9 gene and gene
disruptionissimpleinthatorganism,soitwouldbeagoodalternativemodeltotesthowMTV9
knockoutaffectsplantdevelopmentandphysiologyinthisbasalplant.
3.MTV9localization
Subcellular fractionation of endogenous MTV9 protein and confocal analysis of GFP fusion
constructs suggest that MTV9 localizes primarily at the PVC. MTV9 does not contain a signal
peptideortransmembranedomains, indicatingthatitassociateswiththePVCfromthecytosolic
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side, possibly through post-translationalmodifications or through interactionwith PVC resident
proteins. Recently, a proteomic study identified MTV9 as a putative palmitoylated protein
(Hemsley et al., 2013), suggesting that palmyotylation could mediate its targeting to the PVC.
Palmitoylationisareversiblepost-translationalmodificationthatcovalentlyattachespalmiticacid,
a saturated 16-carbon fatty acid, inmost cases to a cysteine residue. Thismodification confers
hydrophobicitytotheproteinfavoringtheassociationwithmembranes(Leventaletal.,2010).Our
analysis revealedthatMTV9couldonlybeextractedefficiently frommembraneswithdetergent
but not with buffers used to extract peripheralmembrane associated proteins. This pattern of
extraction resembles that of ERBIN, a humanprotein that is recruited to plasmamembrane via
palmitoylationoftwocysteines(Izawaetal.,2008),whichsupportsthatmembraneassociationof
MTV9maybemediatedthroughpalmitoylation.Inthisregard,wehavedeterminedthatthePVC-
targetingmotifofMTV9residesintheC-terminalCTdomain(aminoacids473-678),whichcontains
theonlycysteineresiduepresentinMTV9.WethusproposethatrecruitmentofMTV9tothePVC
maybemediatedbyreversiblepalmitoylationofthatcysteineresidue. Importantly, thecysteine
residueandthenexttwoaminoacidsdefineamotif (CWPmotif) that isstrictlyconserved inall
theMTV9orthologuespresentinplants,suggestingthatitiskeyfortheirfunction.TheCWPmotif
is also present in the STV9 paralogues, which are also localized in endosomal compartments.
Further characterization of the CT domain, including site directed mutagenesis of the CWP
domain,willclarifywhatmotifsarenecessaryforMTV9andSTV9localizationandfunction.Afull
characterizationofthephenotypescausedbyMTV9andSTV9knockoutwillbenecessarytothen
analyzewhatfunctionscanbecomplementedbythedifferentmutantversionsoftheproteins.
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4.MTV9mRNAexpression
Expression studies can provide important hints to uncover gene function. For example,
MTV4/AGD5 is expressed specifically in the petal abscission zone of flowers, and, accordingly,
mtv4 mutants have a nevershed phenotype of delayed petal abscission (Liljegren et al., 2009).
Interestingly, co-expression analysis shows that MTV9 is highly co-expressed with several
traffickinggenes,includingVCL1,VPS39andALIX,whichareinvolvedinPVCtovacuoletransport.
This suggests that coordinated expression of these genes in particular conditions and tissues is
important forproper regulationofvacuolar trafficking.Promoteranalysis supports thatMTV9 is
not expressed ubiquitously throughout the plant, but has a rather specific spatial expression
pattern.Inthematurerootit isexpressedinthevasculatureandinroottipsit isspecificforthe
quiescentcentercells.Inleaves,maximalexpressionisfoundinthevasculatureandintrichomes.
MTV9 is also expressed in the hypocotyl, in the shoots and in the SAM, consistent with the
mutationofMTV9provokingVAC2 secretion in theSAMand leading topremature termination.
Cross sections of shoots reveal highest expression also in the vasculature, indicating thatMTV9
mayhaveafunctionrelatedwithconductivetissues.ThespecificexpressionofMTV9inthesecell
typesandtissueswillinformthesearchforphenotypesinthemtv9knockoutplants.Forinstance,
thevasculatureplaysakeyroleinthetransitiontofloweringbytransmittinginformationfromthe
leaves (the florigen signal, nutritional status, etc.) to the SAM to promote the switch from
vegetativetoreproductivegrowth,soitisinterestingthatmtv9mutantsshowdelayedflowering.
In contrast, no expression ofMTV9was observed in seeds,which is consistentwith the lack of
effectofthemtv9mutationinthosetissues.
99
5.Ontheactivityofthemtv11-1allele
Phosphatidylinositol 3-kinases (PI3Ks) are key enzymes involved inmany aspects of cell biology
andphysiology inorganisms fromalleukaryotickingdoms.ThreeclassesofPI3Ksarepresent in
animalcells,andeachclasshasacharacteristicsubstratespecificityandasetofcellularfunctions.
OnlythePI3KsfromclassIII(PIK3C3class)areconservedacrosskingdoms,andinyeastandplant
genomes they are the sole PI3Ks present. PIK3C3 have an invariant core formed by a catalytic
subunit(VPS34)andaregulatorysubunit(VPS15).ThiscoreinteractswithVPS30,Atg14andAtg38
toformthePIK3C3-C1complexinvolvedinautophagy,withVPS30andVPS38toformthePIK3C3-
C2 complex involved in all the vacuolar trafficking pathways in yeast (biosynthetic, endocytic,
autophagicandthecytoplasm-tovacuolepathways)andwithGPA1andAtg18toformthePIK3C3-
C3 complex involved in pheromone signaling (Reidick et al., 2017). The PIK3C3-C2 complex
synthesizes phosphatidylinositol 3-phosphate (PI3P) at specific domains of the membrane to
recruit effector proteins containing PI3P binding domains, most often PX or FYVE domains,
involvedindifferentstepsofthevacuolartraffickingpathway.UsingthesyntheticbiosensorYFP-
2XFYVEitwasreportedthatPI3PinArabidopsiscellsisgraduallydistributedintheTGN,thePVC
and the tonoplast (Vermeer et al., 2006) consistent with VPS15 and the PI3K functioning
specifically in the vacuolar trafficking pathway. Importantly, themtv11-1 mutation reduces but
does not abolish the accumulation of PI3P in endosomes, indicating that the PI3K activity is
affected but not blocked. Indeed, Arabidopsis null mutants in AtVPS34 or AtVPS15 are
gametophyticlethal(Leeetal.,2008;Wangetal.,2012;Xuetal.,2011).Inthemtv11-1mutantwe
detected abnormally splicedAtVPS15 transcripts encoding two typesof truncatedproteins, one
containing only the N-terminal kinase domain and another containing the N-terminal kinase
domain fused to threeof theC-terminalWDdomains.Most likely, it is this latter fusionprotein
that retains sufficient activity to allow plant viability. Recently, a structure of the PIK3C3-C2
100
complexat4.4Åresolutionwasreported(Rostislavlevaetal.,2015).VPS15andVPS34intertwine
in an antiparallel fashion, with all three domains of VPS15 (Kinase, HEAT and WD domains)
establishing contacts with VPS34. The catalytic domains of both proteins contact each other,
whereas the WD domain of VPS15 bridges the VPS34C2 domain and the VPS30/VPS38 BARA
domains.ThecentralregionofVPS15,whichismissinginthetruncatedmtv11-1protein,isalinker
domainthatestablishesfurthercontactswithVPS34,VPS30andVPS38.Thislinkerdomainadopts
a folded V shape so that the N-terminal kinase domain and the C-terminal WD domains are
actually nearby in the tertiary structure. Hence, the truncated protein could still maintain the
interactionswiththeVPS34kinasedomainatoneendandbridgetheVPS34C2domainandthe
VPS30/VPS38BARAdomainsattheotherend.Thiscouldexplainwhythistruncatedfusionprotein
lackingtheentirecentralregionwouldstillmaintainactivityofthePIK3C3-C2complex.Thestudy
ofthemtv11-1mutantallelemaythenreveal importantinformationonthestructural-functional
relationship of the PIK3C3-C2 complex. The complex containing the truncated AtVPS15 protein
encoded by the mutant allele is probably still correctly targeted to membranes, since the
myristoylationmotif ofAtVPS15 is intact.However, the complexmaybe less stable,becauseof
reduced interaction strength between the subunits. Alternatively, the activity of the catalytic
VPS34 subunit may be compromised. Several catalytic elements of VPS34 contact the VPS15
kinasedomain(Rostislavlevaetal.,2015)sothedeletionofthelinkerdomainmayperturbthese
contactsandaffecttheactivityofVPS34.Analyzinghowthetruncatedproteinreducesbutdoes
notabolishactivityofthePIK3C3-C2complexwilladvanceourunderstandingonhowthiscomplex
synthesizes PI3P on targetmembranes to direct effector recruitment andmembrane trafficking
reactions.
101
6.MTV9andMTV11,traffickingfactorsatthePVC
AlltheMTVgenesisolateduntilnowencodeproteinslocalizedinpost-Golgicompartmentsofthe
biosyntheticpathway.VTI12,AtVPS45,MTV1andMTV4are localizedat theTGNand inclathrin
coatedvesicles (Sanmartinetal.,2007;Saueretal.,2013;Zouharetal.,2009)andMTV2/VSR4,
VSR1andVSR3cyclebetweentheTGNandthePVC(Zouharetal.,2010).MTV9isalsolocalizedat
the PVC,where itmay function in tetheringmembranes for fusion. The compartment(s)where
MTV11 is localized is yet unidentified, but considering that PIP3, the product of the PIK3C3-C2
complex,isenrichedinmembranesoftheTGN,PVCandvacuole,andthatMTV11-GFPdisplaysa
punctatepattern,wecanassumethatMTV11isassociatedmainlywiththeTGNandthePVC.Why
doesthemtvscreenonlyunveilpost-Golgistepsofvacuolartrafficking?Thesestepsarespecific
fortransporttothevacuoleandmutationsaffectingthemshouldnotinterferewithtraffickingto
thePM.Moreover,thepresenceofindependentroutestothevacuoleinplantsmeansthatwhen
one of the pathways is blocked, transport may still proceed through an alternate pathway. In
contrast,mutationsinearlierstepsofthesecretorypathway,attheERortheGolgi,wouldaffect
transportbothtothevacuoleandthePM,andwouldbemoredetrimentaltoplantviability.Early
plant lethality precludes isolation of the mtv mutants, which are detected relatively late in
development. Indeed, some of themtv mutants isolated correspond to hypomorphic alleles of
essential genes that when knocked out cause pollen or embryo lethality. Moreover, the mtv
screenreliesontheabnormalsecretionofVAC2andifbothtransporttothevacuoleandthePM
would be affected, thenVAC2would be retained intracellularly andwould not produce anmtv
phenotype.Thesefeaturesfavortheidentificationofmutantsinthelatesecretorypathway,and
explainswhyallthemutantsisolatedthusfarareingenesactinginpost-Golgicompartments.
102
7.Arsenatephytoremediation
Although in standard growth media or in soil the mtv11-1 shows a growth delay, in media
containing high As(V) concentration,mtv11-1 has significantly increased growth relative to Wt
plants,suggestingthatitismoretoleranttothistoxicmetalloid.Themtv11-1mutantshowsclear
accumulation of the PM PHT1;1 high affinity phosphate (and As(V)) transporter in endocytic
organelles.This retention inendosomes isconsistentwithvacuolar transportbeingperturbed in
themutant.Thislikelyimpairsproperturnoverofthetransporterinthevacuoleandmayincrease
their recycling into the PM. Indeed, we found increased levels of PHT1;1 in the PM in media
containing As(V), which normally induces the internalization and vacuolar degradation of these
phosphate transporters (Castrilloetal.,2013).The failure to recycleproperly these transporters
into thevacuoleand theiraccumulation in thePM implies that themtv11-1mutantmay in fact
uptakemoreAs(V)thanWtplants,butstillbemoretoleranttothistoxicmetalloid.Ifindeedthis
were the case, understanding how themtv11-1mutant achieves As(V) hyper-accumulation and
higher tolerancewouldbeofgreat interest for thebioremediation field.Apossibility is that the
alterationsinthevacuolarpathwayallowforhigherlevelsofAs(III)sequestrationinthevacuole.
Measuring As(V) and As(III) levels in whole tissues and in isolated vacuoles (Zouhar, 2017) will
clarify if themtv11-1mutanthyper-accumulatesAs(V)andhas increasedvacuolar sequestration
capacity forAs(III). Itwill also be interesting to analyze if the reduced activity of thePIK3C3-C2
complexdirectly results inhigherAs(V) tolerance. In thatcase thiscomplexcouldbea target to
breedplantswithimprovedphytoremediationproperties.
103
Conclusions
1) Theanalysisoffourindependentmtv9allelesindicatesthatMTV9isessentialforvacuolartraffickingofVAC2invegetativetissues.
2) MTV9encodesacoiled-coilproteinthathomodimerizes.
3) MTV9islocalizedprimarilyattheprevacuolarcompartment.
4) MTV9overexpressionaltersPVCmorphologyandblocksvacuolartraffickingbutnottransporttotheplasmamembrane.
5) TheCTdomainofMTV9containsthePVCtargetinginformation.
6) Themtv11mutantphenotypeiscausedbyapointmutationinaspliceacceptorsiteofthe7thintronoftheAtVPS15gene.
7) Thehypomorphicmtv11-1alleleaccumulatesalternativelysplicedAtVPS15transcripts,includingatranscriptthatskipsexon8andretainstheopenreadingframe.
8) TheactivityoftheAtVPS15isrequiredfortraffickingofendogenousvacuolarstorageproteinsinseedtissues.
9) Themtv11-1mutationcausesreducedPIP3levelsinendosomes.
10) Themtv11-1mutantretainsmorePHT1;1intheplasmamembranebutismoretoleranttoAs(V)inthemedia,suggestingthatitcombinestolerancewithhyperaccumulation.
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